Methods and compositions for producing hydrocarbons

ABSTRACT

Compositions and methods for producing aldehydes, alkanes, and alkenes are described herein. The aldehydes, alkanes, and alkenes can be used in biofuels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/625,107,filed Sep. 24, 2012, which is a continuation of U.S. Pat. No. 8,323,924,filed Feb. 22, 2010, which is a continuation-in-part of InternationalApplication No. PCT/US09/44403, filed May 18, 2009, which claims thebenefit of U.S. Provisional Application No. 61/053,955, filed May 16,2008, the contents of all of which are hereby incorporated by referencein their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 148,986 byte ASCII (Text) file named“LS00012 PCT_SeqListing_12.05.10” created on Dec. 5, 2010.

BACKGROUND OF THE INVENTION

Petroleum is a limited, natural resource found in the Earth in liquid,gaseous, or solid forms. Petroleum is primarily composed ofhydrocarbons, which are comprised mainly of carbon and hydrogen. It alsocontains significant amounts of other elements, such as, nitrogen,oxygen, or sulfur, in different forms.

Petroleum is a valuable resource, but petroleum products are developedat considerable costs, both financial and environmental. First, sourcesof petroleum must be discovered. Petroleum exploration is an expensiveand risky venture. The cost of exploring deep water wells can exceed$100 million. Moreover, there is no guarantee that these wells willcontain petroleum. It is estimated that only 40% of drilled wells leadto productive wells generating commercial hydrocarbons. In addition tothe economic cost, petroleum exploration carries a high environmentalcost. For example, offshore exploration disturbs the surrounding marineenvironments.

After a productive well is discovered, the petroleum must be extractedfrom the Earth at great expense. During primary recovery, the naturalpressure underground is sufficient to extract about 20% of the petroleumin the well. As this natural pressure falls, secondary recovery methodsare employed, if economical. Generally, secondary recovery involvesincreasing the well's pressure by, for example, water injection, naturalgas injection, or gas lift. Using secondary recovery methods, anadditional 5% to 15% of petroleum is recovered. Once secondary recoverymethods are exhausted, tertiary recovery methods can be used, ifeconomical. Tertiary methods involve reducing the viscosity of thepetroleum to make it easier to extract. Using tertiary recovery methods,an additional 5% to 15% of petroleum is recovered. Hence, even under thebest circumstances, only 50% of the petroleum in a well can beextracted. Petroleum extraction also carries an environmental cost. Forexample, petroleum extraction can result in large seepages of petroleumrising to the surface. Moreover, offshore drilling involves dredging theseabed which disrupts or destroys the surrounding marine environment.

Since petroleum deposits are not found uniformly throughout the Earth,petroleum must be transported over great distances from petroleumproducing regions to petroleum consuming regions. In addition to theshipping costs, there is also the environmental risk of devastating oilspills.

In its natural form, crude petroleum extracted from the Earth has fewcommercial uses. It is a mixture of hydrocarbons (e.g., paraffins (oralkanes), olefins (or alkenes), alkynes, napthenes (or cylcoalkanes),aliphatic compounds, aromatic compounds, etc.) of varying length andcomplexity. In addition, crude petroleum contains other organiccompounds (e.g., organic compounds containing nitrogen, oxygen, sulfur,etc.) and impurities (e.g., sulfur, salt, acid, metals, etc.).

Hence, crude petroleum must be refined and purified before it can beused commercially. Due to its high energy density and its easytransportability, most petroleum is refined into fuels, such astransportation fuels (e.g., gasoline, diesel, aviation fuel, etc.),heating oil, liquefied petroleum gas, etc.

Crude petroleum is also a primary source of raw materials for producingpetrochemicals. The two main classes of raw materials derived frompetroleum are short chain olefins (e.g., ethylene and propylene) andaromatics (e.g., benzene and xylene isomers). These raw materials arederived from longer chain hydrocarbons in crude petroleum by cracking itat considerable expense using a variety of methods, such as catalyticcracking, steam cracking, or catalytic reforming. These raw materialsare used to make petrochemicals, which cannot be directly refined fromcrude petroleum, such as monomers, solvents, detergents, or adhesives.

One example of a raw material derived from crude petroleum is ethylene.Ethylene is used to produce petrochemicals such as, polyethylene,ethanol, ethylene oxide, ethylene glycol, polyester, glycol ether,ethoxylate, vinyl acetate, 1,2-dichloroethane, trichloroethylene,tetrachloroethylene, vinyl chloride, and polyvinyl chloride. Anadditional example of a raw material is propylene, which is used toproduce isopropyl alcohol, acrylonitrile, polypropylene, propyleneoxide, propylene glycol, glycol ethers, butylene, isobutylene,1,3-butadiene, synthetic elastomers, polyolefins, alpha-olefins, fattyalcohols, acrylic acid, acrylic polymers, allyl chloride,epichlorohydrin, and epoxy resins.

These petrochemicals can then be used to make specialty chemicals, suchas plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, orgels. Particular specialty chemicals which can be produced frompetrochemical raw materials are: fatty acids, hydrocarbons (e.g., longchain, branched chain, saturated, unsaturated, etc.), fatty alcohols,esters, fatty aldehydes, ketones, lubricants, etc.

Specialty chemicals have many commercial uses. Fatty acids are usedcommercially as surfactants, for example, in detergents and soaps. Theycan also be used as additives in fuels, lubricating oils, paints,lacquers, candles, salad oil, shortening, cosmetics, and emulsifiers. Inaddition, fatty acids are used as accelerator activators in rubberproducts. Fatty acids can also be used as a feedstock to produce methylesters, amides, amines, acid chlorides, anhydrides, ketene dimers, andperoxy acids and esters.

Hydrocarbons have many commercial uses. For example, shorter chainalkanes are used as fuels. Methane and ethane are the main constituentsof natural gas. Longer chain alkanes (e.g., from five to sixteencarbons) are used as transportation fuels (e.g., gasoline, diesel, oraviation fuel). Alkanes having more than sixteen carbon atoms areimportant components of fuel oils and lubricating oils. Even longeralkanes, which are solid at room temperature, can be used, for example,as a paraffin wax. Alkanes that contain approximately thirty-fivecarbons are found in bitumen, which is used for road surfacing. Inaddition, longer chain alkanes can be cracked to produce commerciallyuseful shorter chain hydrocarbons.

Like short chain alkanes, short chain alkenes are used in transportationfuels. Longer chain alkenes are used in plastics, lubricants, andsynthetic lubricants. In addition, alkenes are used as a feedstock toproduce alcohols, esters, plasticizers, surfactants, tertiary amines,enhanced oil recovery agents, fatty acids, thiols, alkenylsuccinicanhydrides, epoxides, chlorinated alkanes, chlorinated alkenes, waxes,fuel additives, and drag flow reducers.

Fatty alcohols have many commercial uses. The shorter chain fattyalcohols are used in the cosmetic and food industries as emulsifiers,emollients, and thickeners. Due to their amphiphilic nature, fattyalcohols behave as nonionic surfactants, which are useful as detergents.In addition, fatty alcohols are used in waxes, gums, resins,pharmaceutical salves and lotions, lubricating oil additives, textileantistatic and finishing agents, plasticizers, cosmetics, industrialsolvents, and solvents for fats.

Esters have many commercial uses. For example, biodiesel, an alternativefuel, is comprised of esters (e.g., fatty acid methyl ester, fatty acidethyl esters, etc.). Some low molecular weight esters are volatile witha pleasant odor which makes them useful as fragrances or flavoringagents. In addition, esters are used as solvents for lacquers, paints,and varnishes. Furthermore, some naturally occurring substances, such aswaxes, fats, and oils are comprised of esters. Esters are also used assoftening agents in resins and plastics, plasticizers, flame retardants,and additives in gasoline and oil. In addition, esters can be used inthe manufacture of polymers, films, textiles, dyes, and pharmaceuticals.

Aldehydes are used to produce many specialty chemicals. For example,aldehydes are used to produce polymers, resins (e.g., Bakelite), dyes,flavorings, plasticizers, perfumes, pharmaceuticals, and otherchemicals. Some are used as solvents, preservatives, or disinfectants.Some natural and synthetic compounds, such as vitamins and hormones, arealdehydes. In addition, many sugars contain aldehyde groups.

Ketones are used commercially as solvents. For example, acetone isfrequently used as a solvent, but it is also a raw material for makingpolymers. Ketones are also used in lacquers, paints, explosives,perfumes, and textile processing. In addition, ketones are used toproduce alcohols, alkenes, alkanes, imines, and enamines.

In addition, crude petroleum is a source of lubricants. Lubricantsderived petroleum are typically composed of olefins, particularlypolyolefins and alpha-olefins. Lubricants can either be refined fromcrude petroleum or manufactured using raw materials refined from crudepetroleum.

Obtaining these specialty chemicals from crude petroleum requires asignificant financial investment as well as a great deal of energy. Itis also an inefficient process because frequently the long chainhydrocarbons in crude petroleum are cracked to produce smaller monomers.These monomer are then used as the raw material to manufacture the morecomplex specialty chemicals.

In addition to the problems with exploring, extracting, transporting,and refining petroleum, petroleum is a limited and dwindling resource.One estimate of world petroleum consumption is 30 billion barrels peryear. By some estimates, it is predicted that at current productionlevels, the world's petroleum reserves could be depleted before the year2050.

Finally, the burning of petroleum based fuels releases greenhouse gases(e.g., carbon dioxide) and other forms of air pollution (e.g., carbonmonoxide, sulfur dioxide, etc.). As the world's demand for fuelincreases, the emission of greenhouse gases and other forms of airpollution also increases. The accumulation of greenhouse gases in theatmosphere leads to an increase global warming. Hence, in addition todamaging the environment locally (e.g., oil spills, dredging of marineenvironments, etc.), burning petroleum also damages the environmentglobally.

Due to the inherent challenges posed by petroleum, there is a need for arenewable petroleum source which does not need to be explored,extracted, transported over long distances, or substantially refinedlike petroleum. There is also a need for a renewable petroleum sourcethat can be produced economically without creating the type ofenvironmental damage produced by the petroleum industry and the burningof petroleum based fuels. For similar reasons, there is also a need fora renewable source of chemicals that are typically derived frompetroleum.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the identification ofcyanobacterial genes that encode hydrocarbon biosynthetic polypeptides.Accordingly, in one aspect, the invention features a method of producinga hydrocarbon, the method comprising producing in a host cell apolypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, or a variantthereof, and isolating the hydrocarbon from the host cell.

In some embodiments, the polypeptide comprises an amino acid sequencehaving at least about 70%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% identity toSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, or 36.

In some embodiments, the polypeptide comprises the amino acid sequenceof SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, or 36 with one or more amino acid substitutions, additions,insertions, or deletions. In some embodiments, the polypeptide hasdecarbonylase activity. In yet other embodiments, the polypeptidecomprises the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, with one or moreconservative amino acid substitutions. For example, the polypeptidecomprises one or more of the following conservative amino acidsubstitutions: replacement of an aliphatic amino acid, such as alanine,valine, leucine, and isoleucine, with another aliphatic amino acid;replacement of a serine with a threonine; replacement of a threoninewith a serine; replacement of an acidic residue, such as aspartic acidand glutamic acid, with another acidic residue; replacement of a residuebearing an amide group, such as asparagine and glutamine, with anotherresidue bearing an amide group; exchange of a basic residue, such aslysine and arginine, with another basic residue; and replacement of anaromatic residue, such as phenylalanine and tyrosine, with anotheraromatic residue. In some embodiments, the polypeptide has about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, ormore amino acid substitutions, additions, insertions, or deletions. Insome embodiments, the polypeptide has decarbonylase activity.

In other embodiments, the polypeptide comprises the amino acid sequenceof: (i) SEQ ID NO:37 or SEQ ID NO:38 or SEQ ID NO:39; or (ii) SEQ IDNO:40 and any one of (a) SEQ ID NO:37, (b) SEQ ID NO:38, and (c) SEQ IDNO:39; or (iii) SEQ ID NO:41 or SEQ ID NO:42 or SEQ ID NO:43 or SEQ IDNO:44. In certain embodiments, the polypeptide has decarbonylaseactivity.

In another aspect, the invention features a method of producing ahydrocarbon, the method comprising expressing in a host cell apolynucleotide comprising a nucleotide sequence having at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, or 35. In some embodiments, the nucleotide sequence is SEQID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or35. In some embodiments, the method further comprises isolating thehydrocarbon from the host cell.

In other embodiments, the nucleotide sequence hybridizes to a complementof SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, or 35, or to a fragment thereof, for example, under low stringency,medium stringency, high stringency, or very high stringency conditions.

In other embodiments, the nucleotide sequence encodes a polypeptidecomprising: (i) the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36; or (ii) the aminoacid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, or 36 with one or more amino acid substitutions,additions, insertions, or deletions. In some embodiments, thepolypeptide comprises the amino acid sequence of SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 with one ormore conservative amino acid substitutions. In some embodiments, thepolypeptide has decarbonylase activity.

In other embodiments, the nucleotide sequence encodes a polypeptidehaving the same biological activity as a polypeptide comprising theamino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, or 36. In some embodiments, the nucleotidesequence is SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, or 35 or a fragment thereof. In other embodiments, thenucleotide sequence hybridizes to a complement of SEQ ID NO:1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35 or to afragment thereof, for example, under low stringency, medium stringency,high stringency, or very high stringency conditions. In someembodiments, the biological activity is decarbonylase activity.

In some embodiments, the method comprises transforming a host cell witha recombinant vector comprising a nucleotide sequence having at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% sequenceidentity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, or 35. In some embodiments, the recombinant vector furthercomprises a promoter operably linked to the nucleotide sequence. In someembodiments, the promoter is a developmentally-regulated, anorganelle-specific, a tissue-specific, an inducible, a constitutive, ora cell-specific promoter. In particular embodiments, the recombinantvector comprises at least one sequence selected from the groupconsisting of (a) a regulatory sequence operatively coupled to thenucleotide sequence; (b) a selection marker operatively coupled to thenucleotide sequence; (c) a marker sequence operatively coupled to thenucleotide sequence; (d) a purification moiety operatively coupled tothe nucleotide sequence; (e) a secretion sequence operatively coupled tothe nucleotide sequence; and (f) a targeting sequence operativelycoupled to the nucleotide sequence. In certain embodiments, thenucleotide sequence is stably incorporated into the genomic DNA of thehost cell, and the expression of the nucleotide sequence is under thecontrol of a regulated promoter region.

In any of the aspects described herein, the host cell can be selectedfrom the group consisting of a mammalian cell, plant cell, insect cell,yeast cell, fungus cell, filamentous fungi cell, and bacterial cell.

In some embodiments, the host cell is a Gram-positive bacterial cell. Inother embodiments, the host cell is a Gram-negative bacterial cell.

In some embodiments, the host cell is selected from the genusEscherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas,Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor,Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete,Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas,Schizosaccharomyces, Yarrowia, or Streptomyces.

In particular embodiments, the host cell is a Bacillus lentus cell, aBacillus brevis cell, a Bacillus stearothermophilus cell, a Bacilluslicheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulanscell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillusthuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell,a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.

In other embodiments, the host cell is a Trichoderma koningii cell, aTrichoderma viride cell, a Trichoderma reesei cell, a Trichodermalongibrachiatum cell, an Aspergillus awamori cell, an Aspergillusfumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulanscell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicolainsolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, aRhizomucor miehei cell, or a Mucor michei cell.

In yet other embodiments, the host cell is a Streptomyces lividans cellor a Streptomyces murinus cell. In other embodiments, the host cell isan Actinomycetes cell.

In some embodiments, the host cell is a CHO cell, a COS cell, a VEROcell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a3T3 cell, or a PC12 cell.

In particular embodiments, the host cell is an E. coli cell, such as astrain B, a strain C, a strain K, or a strain W E. coli cell.

In other embodiments, the host cell is a cyanobacterial host cell. Inparticular embodiments, the cyanobacterial host cell is a cell listed inTable 1.

In some embodiments, the hydrocarbon is secreted from by the host cell.

In certain embodiments, the host cell overexpresses a substratedescribed herein. In some embodiments, the method further includestransforming the host cell with a nucleic acid that encodes an enzymedescribed herein, and the host cell overexpresses a substrate describedherein. In other embodiments, the method further includes culturing thehost cell in the presence of at least one substrate described herein. Insome embodiments, the substrate is a fatty acid derivative, an acyl-ACP,a fatty acid, an acyl-CoA, a fatty aldehyde, a fatty alcohol, or a fattyester.

In some embodiments, the fatty acid derivative substrate is anunsaturated fatty acid derivative substrate, a monounsaturated fattyacid derivative substrate, or a saturated fatty acid derivativesubstrate. In other embodiments, the fatty acid derivative substrate isa straight chain fatty acid derivative substrate, a branched chain fattyacid derivative substrate, or a fatty acid derivative substrate thatincludes a cyclic moiety.

In certain embodiments of the aspects described herein, the hydrocarbonproduced is an alkane. In some embodiments, the alkane is a C₃-C₂₅alkane. For example, the alkane is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, orC₂₅ alkane. In some embodiments, the alkane is tridecane,methyltridecane, nonadecane, methylnonadecane, heptadecane,methylheptadecane, pentadecane, or methylpentadecane.

In some embodiments, the alkane is a straight chain alkane, a branchedchain alkane, or a cyclic alkane.

In certain embodiments, the method further comprises culturing the hostcell in the presence of a saturated fatty acid derivative, and thehydrocarbon produced is an alkane. In certain embodiments, the saturatedfatty acid derivative is a C₆-C₂₆ fatty acid derivative substrate. Forexample, the fatty acid derivative substrate is a C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄,C₂₅, or a C₂₆ fatty acid derivative substrate. In particularembodiments, the fatty acid derivative substrate is 2-methylicosanal,icosanal, octadecanal, tetradecanal, 2-methyloctadecanal, stearaldehyde,or palmitaldehyde.

In some embodiments, the method further includes isolating the alkanefrom the host cell or from the culture medium. In other embodiments, themethod further includes cracking or refining the alkane.

In certain embodiments of the aspects described herein, the hydrocarbonproduced is an alkene. In some embodiments, the alkene is a C₃-C₂₅alkene. For example, the alkene is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, orC₂₅ alkene. In some embodiments, the alkene is pentadecene, heptadecene,methylpentadecene, or methylheptadecene.

In some embodiments, the alkene is a straight chain alkene, a branchedchain alkene, or a cyclic alkene.

In certain embodiments, the method further comprises culturing the hostcell in the presence of an unsaturated fatty acid derivative, and thehydrocarbon produced is an alkene. In certain embodiments, theunsaturated fatty acid derivative is a C₆-C₂₆ fatty acid derivativesubstrate. For example, the fatty acid derivative substrate is a C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂,C₂₃, C₂₄, C₂₅, or a C₂₆ unsaturated fatty acid derivative substrate. Inparticular embodiments, the fatty acid derivative substrate isoctadecenal, hexadecenal, methylhexadecenal, or methyloctadecenal.

In another aspect, the invention features a genetically engineeredmicroorganism comprising an exogenous control sequence stablyincorporated into the genomic DNA of the microorganism. In oneembodiment, the control sequence is integrated upstream of apolynucleotide comprising a nucleotide sequence having at least about70% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, or 35. In some embodiments, the nucleotidesequence has at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% sequenceidentity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, or 35. In some embodiments, the nucleotide sequence is SEQID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or35.

In some embodiments, the polynucleotide is endogenous to themicroorganism. In some embodiments, the microorganism expresses anincreased level of a hydrocarbon relative to a wild-type microorganism.In some embodiments, the microorganism is a cyanobacterium.

In another aspect, the invention features a method of making ahydrocarbon, the method comprising culturing a genetically engineeredmicroorganism described herein under conditions suitable for geneexpression, and isolating the hydrocarbon.

In another aspect, the invention features a method of making ahydrocarbon, comprising contacting a substrate with (i) a polypeptidehaving at least 70% identity to the amino acid sequence of SEQ ID NO:2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, or avariant thereof; (ii) a polypeptide encoded by a nucleotide sequencehaving at least 70% identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, or 35, or a variant thereof; (iii) apolypeptide comprising the amino acid sequence of SEQ ID NO:37, 38, or39; (iv) a polypeptide comparing the amino acid sequence of SEQ ID NO:40and any one of (a) SEQ ID NO:37, (b) SEQ ID NO:38, and (c) SEQ ID NO:39;or (v) SEQ ID NO:41, 42, 43, or 44. In some embodiments, the polypeptidehas decarbonylase activity.

In some embodiments, the polypeptide has at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, or 36. In some embodiments, the polypeptide has theamino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, or 36.

In some embodiments, the polypeptide is encoded by a nucleotide sequencehaving at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, or 35. In some embodiments, the polypeptide is encoded by anucleotide sequence having SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, or 35.

In some embodiments, the biological substrate is a fatty acidderivative, an acyl-ACP, a fatty acid, an acyl-CoA, a fatty aldehyde, afatty alcohol, or a fatty ester.

In some embodiments, the substrate is a saturated fatty acid derivative,and the hydrocarbon is an alkane, for example, a C₃-C₂₅ alkane. Forexample, the alkane is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃,C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, or C₂₅ alkane. Insome embodiments, the alkane is tridecane, methyltridecane, nonadecane,methylnonadecane, heptadecane, methylheptadecane, pentadecane, ormethylpentadecane.

In some embodiments, the alkane is a straight chain alkane, a branchedchain alkane, or a cyclic alkane.

In some embodiments, the saturated fatty acid derivative is2-methylicosanal, icosanal, octadecanal, tetradecanal,2-methyloctadecanal, stearaldehyde, or palmitaldehyde.

In other embodiments, the biological substrate is an unsaturated fattyacid derivative, and the hydrocarbon is an alkene, for example, a C₃-C₂₅alkene. For example, the alkene is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, orC₂₅ alkene. In some embodiments, the alkene is pentadecene, heptadecene,methylpentadecene, or methylheptadecene.

In some embodiments, the alkene is a straight chain alkene, a branchedchain alkene, or a cyclic alkene.

In some embodiments, the unsaturated fatty acid derivative isoctadecenal, hexadecenal, methylhexadecenal, or methyloctadecenal.

In another aspect, the invention features a hydrocarbon produced by anyof the methods or microorganisms described herein. In particularembodiments, the hydrocarbon is an alkane or an alkene having a δ¹³C ofabout −15.4 or greater. For example, the alkane or alkene has a δ¹³C ofabout −15.4 to about −10.9, for example, about −13.92 to about −13.84.In other embodiments, the alkane or alkene has an f_(M) ¹⁴C of at leastabout 1.003. For example, the alkane or alkene has an f_(M) ¹⁴C of atleast about 1.01 or at least about 1.5. In some embodiments, the alkaneor alkene has an f_(M) ¹⁴C of about 1.111 to about 1.124.

In another aspect, the invention features a biofuel that includes ahydrocarbon produced by any of the methods or microorganisms describedherein. In particular embodiments, the hydrocarbon is an alkane oralkene having a δ¹³C of about −15.4 or greater. For example, the alkaneor alkene has a δ¹³C of about −15.4 to about −10.9, for example, about−13.92 to about −13.84. In other embodiments, the alkane or alkene hasan f_(M) ¹⁴C of at least about 1.003. For example, the alkane or alkenehas an f_(M) ¹⁴C of at least about 1.01 or at least about 1.5. In someembodiments, the alkane or alkene has an f_(M) ¹⁴C of about 1.111 toabout 1.124. In some embodiments, the biofuel is diesel, gasoline, orjet fuel.

In another aspect, the invention features an isolated nucleic acidconsisting of no more than about 500 nucleotides of SEQ ID NO:1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In someembodiments, the nucleic acid consists of no more than about 300nucleotides, no more than about 350 nucleotides, no more than about 400nucleotides, no more than about 450 nucleotides, no more than about 550nucleotides, no more than about 600 nucleotides, or no more than about650 nucleotides, of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, or 35. In some embodiments, the nucleic acid encodesa polypeptide having decarbonylase activity.

In another aspect, the invention features an isolated nucleic acidconsisting of no more than about 99%, no more than about 98%, no morethan about 97%, no more than about 96%, no more than about 95%, no morethan about 94%, no more than about 93%, no more than about 92%, no morethan about 91%, no more than about 90%, no more than about 85%, or nomore than about 80% of the nucleotides of SEQ ID NO:1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In some embodiments,the nucleic acid encodes a polypeptide having decarbonylase activity.

In another aspect, the invention features an isolated polypeptideconsisting of no more than about 200, no more than about 175, no morethan about 150, or no more than about 100 of the amino acids of SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or36. In some embodiments, the polypeptide has decarbonylase activity.

In another aspect, the invention features an isolated polypeptideconsisting of no more than about 99%, no more than about 98%, no morethan about 97%, no more than about 96%, no more than about 95%, no morethan about 94%, no more than about 93%, no more than about 92%, no morethan about 91%, no more than about 90%, no more than about 85%, or nomore than about 80% of the amino acids of SEQ ID NO:2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some embodiments,the polypeptide has decarbonylase activity.

Definitions

Throughout the specification, a reference may be made using anabbreviated gene name or polypeptide name, but it is understood thatsuch an abbreviated gene or polypeptide name represents the genus ofgenes or polypeptides. Such gene names include all genes encoding thesame polypeptide and homologous polypeptides having the samephysiological function. Polypeptide names include all polypeptides thathave the same activity (e.g., that catalyze the same fundamentalchemical reaction).

The accession numbers referenced herein are derived from the NCBIdatabase (National Center for Biotechnology Information) maintained bythe National Institute of Health, U.S.A. Unless otherwise indicated, theaccession numbers are as provided in the database as of April 2009.

EC numbers are established by the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (NC-IUBMB)(available at http://www.chem.qmul.ac.uk/iubmb/enzyme/). The EC numbersreferenced herein are derived from the KEGG Ligand database, maintainedby the Kyoto Encyclopedia of Genes and Genomics, sponsored in part bythe University of Tokyo. Unless otherwise indicated, the EC numbers areas provided in the database as of March 2008.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” is used herein to mean a value±20% of a given numericalvalue. Thus, “about 60%” means a value of between 60±(20% of 60) (i.e.,between 48 and 70).

As used herein, the term “aldehyde” means a hydrocarbon having theformula RCHO characterized by an unsaturated carbonyl group (C═O). In apreferred embodiment, the aldehyde is any aldehyde made from a fattyacid or fatty acid derivative. In one embodiment, the R group is atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 carbons in length.

As used herein, an “aldehyde biosynthetic gene” or an “aldehydebiosynthetic polynucleotide” is a nucleic acid that encodes an aldehydebiosynthetic polypeptide.

As used herein, an “aldehyde biosynthetic polypeptide” is a polypeptidethat is a part of the biosynthetic pathway of an aldehyde. Suchpolypeptides can act on a biological substrate to yield an aldehyde. Insome instances, the aldehyde biosynthetic polypeptide has reductaseactivity.

As used herein, the term “alkane” means a hydrocarbon containing onlysingle carbon-carbon bonds.

As used herein, an “alkane biosynthetic gene” or an “alkane biosyntheticpolynucleotide” is a nucleic acid that encodes an alkane biosyntheticpolypeptide.

As used herein, an “alkane biosynthetic polypeptide” is a polypeptidethat is a part of the biosynthetic pathway of an alkane. Suchpolypeptides can act on a biological substrate to yield an alkane. Insome instances, the alkane biosynthetic polypeptide has decarbonylaseactivity.

As used herein, an “alkene biosynthetic gene” or an “alkene biosyntheticpolynucleotide” is a nucleic acid that encodes an alkene biosyntheticpolypeptide.

As used herein, an “alkene biosynthetic polypeptide” is a polypeptidethat is a part of the biosynthetic pathway of an alkene. Suchpolypeptides can act on a biological substrate to yield an alkene. Insome instances, the alkene biosynthetic polypeptide has decarbonylaseactivity.

As used herein, the term “attenuate” means to weaken, reduce ordiminish. For example, a polypeptide can be attenuated by modifying thepolypeptide to reduce its activity (e.g., by modifying a nucleotidesequence that encodes the polypeptide).

As used herein, the term “biodiesel” means a biofuel that can be asubstitute of diesel, which is derived from petroleum. Biodiesel can beused in internal combustion diesel engines in either a pure form, whichis referred to as “neat” biodiesel, or as a mixture in any concentrationwith petroleum-based diesel. Biodiesel can include esters orhydrocarbons, such as aldehydes and alkanes.

As used therein, the term “biofuel” refers to any fuel derived frombiomass. Biofuels can be substituted for petroleum based fuels. Forexample, biofuels are inclusive of transportation fuels (e.g., gasoline,diesel, jet fuel, etc.), heating fuels, and electricity-generatingfuels. Biofuels are a renewable energy source.

As used herein, the term “biomass” refers to a carbon source derivedfrom biological material. Biomass can be converted into a biofuel. Oneexemplary source of biomass is plant matter. For example, corn, sugarcane, or switchgrass can be used as biomass. Another non-limitingexample of biomass is animal matter, for example cow manure. Biomassalso includes waste products from industry, agriculture, forestry, andhouseholds. Examples of such waste products that can be used as biomassare fermentation waste, straw, lumber, sewage, garbage, and foodleftovers. Biomass also includes sources of carbon, such ascarbohydrates (e.g., monosaccharides, disaccharides, orpolysaccharides).

As used herein, the phrase “carbon source” refers to a substrate orcompound suitable to be used as a source of carbon for prokaryotic orsimple eukaryotic cell growth. Carbon sources can be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO₂).These include, for example, various monosaccharides, such as glucose,fructose, mannose, and galactose; oligosaccharides, such asfructo-oligosaccharide and galacto-oligosaccharide; polysaccharides suchas xylose and arabinose; disaccharides, such as sucrose, maltose, andturanose; cellulosic material, such as methyl cellulose and sodiumcarboxymethyl cellulose; saturated or unsaturated fatty acid esters,such as succinate, lactate, and acetate; alcohols, such as ethanol ormixtures thereof. The carbon source can also be a product ofphotosynthesis, including, but not limited to, glucose. A preferredcarbon source is biomass. Another preferred carbon source is glucose.

As used herein, a “cloud point lowering additive” is an additive addedto a composition to decrease or lower the cloud point of a solution.

As used herein, the phrase “cloud point of a fluid” means thetemperature at which dissolved solids are no longer completely soluble.Below this temperature, solids begin precipitating as a second phasegiving the fluid a cloudy appearance. In the petroleum industry, cloudpoint refers to the temperature below which a solidified material orother heavy hydrocarbon crystallizes in a crude oil, refined oil, orfuel to form a cloudy appearance. The presence of solidified materialsinfluences the flowing behavior of the fluid, the tendency of the fluidto clog fuel filters, injectors, etc., the accumulation of solidifiedmaterials on cold surfaces (e.g., a pipeline or heat exchanger fouling),and the emulsion characteristics of the fluid with water.

A nucleotide sequence is “complementary” to another nucleotide sequenceif each of the bases of the two sequences matches (i.e., is capable offorming Watson Crick base pairs). The term “complementary strand” isused herein interchangeably with the term “complement”. The complementof a nucleic acid strand can be the complement of a coding strand or thecomplement of a non-coding strand.

As used herein, the term “conditions sufficient to allow expression”means any conditions that allow a host cell to produce a desiredproduct, such as a polypeptide, aldehyde, or alkane described herein.Suitable conditions include, for example, fermentation conditions.Fermentation conditions can comprise many parameters, such astemperature ranges, levels of aeration, and media composition. Each ofthese conditions, individually and in combination, allows the host cellto grow. Exemplary culture media include broths or gels. Generally, themedium includes a carbon source, such as glucose, fructose, cellulose,or the like, that can be metabolized by a host cell directly. Inaddition, enzymes can be used in the medium to facilitate themobilization (e.g., the depolymerization of starch or cellulose tofermentable sugars) and subsequent metabolism of the carbon source.

To determine if conditions are sufficient to allow expression, a hostcell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48hours. During and/or after culturing, samples can be obtained andanalyzed to determine if the conditions allow expression. For example,the host cells in the sample or the medium in which the host cells weregrown can be tested for the presence of a desired product. When testingfor the presence of a product, assays, such as, but not limited to, TLC,HPLC, GC/FID, GC/MS, LC/MS, MS, can be used.

It is understood that the polypeptides described herein may haveadditional conservative or non-essential amino acid substitutions, whichdo not have a substantial effect on the polypeptide functions. Whetheror not a particular substitution will be tolerated (i.e., will notadversely affect desired biological properties, such as decarboxylaseactivity) can be determined as described in Bowie et al., Science (1990)247:1306 1310. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

As used herein, “control element” means a transcriptional controlelement. Control elements include promoters and enhancers. The term“promoter element,” “promoter,” or “promoter sequence” refers to a DNAsequence that functions as a switch that activates the expression of agene. If the gene is activated, it is said to be transcribed orparticipating in transcription. Transcription involves the synthesis ofmRNA from the gene. A promoter, therefore, serves as a transcriptionalregulatory element and also provides a site for initiation oftranscription of the gene into mRNA. Control elements interactspecifically with cellular proteins involved in transcription (Maniatiset al., Science 236:1237, 1987).

As used herein, the term “ester synthase” means a peptide capable ofproducing fatty esters. More specifically, an ester synthase is apeptide which converts a thioester to a fatty ester. In a preferredembodiment, the ester synthase converts a thioester (e.g., acyl-CoA) toa fatty ester.

In an alternate embodiment, an ester synthase uses a thioester and analcohol as substrates to produce a fatty ester. Ester synthases arecapable of using short and long chain thioesters as substrates. Inaddition, ester synthases are capable of using short and long chainalcohols as substrates.

Non-limiting examples of ester synthases are wax synthases, wax-estersynthases, acyl CoA:alcohol transacylases, acyltransferases, and fattyacyl-coenzyme A:fatty alcohol acyltransferases. Exemplary estersynthases are classified in enzyme classification number EC 2.3.1.75.Exemplary GenBank Accession Numbers are provided in FIG. 40.

As used herein, the term “fatty acid” means a carboxylic acid having theformula RCOOH. R represents an aliphatic group, preferably an alkylgroup. R can comprise between about 4 and about 22 carbon atoms. Fattyacids can be saturated, monounsaturated, or polyunsaturated. In apreferred embodiment, the fatty acid is made from a fatty acidbiosynthetic pathway.

As used herein, the term “fatty acid biosynthetic pathway” means abiosynthetic pathway that produces fatty acids. The fatty acidbiosynthetic pathway includes fatty acid enzymes that can be engineered,as described herein, to produce fatty acids, and in some embodiments canbe expressed with additional enzymes to produce fatty acids havingdesired carbon chain characteristics.

As used herein, the term “fatty acid derivative” means products made inpart from the fatty acid biosynthetic pathway of the production hostorganism. “Fatty acid derivative” also includes products made in partfrom acyl-ACP or acyl-ACP derivatives. The fatty acid biosyntheticpathway includes fatty acid synthase enzymes which can be engineered asdescribed herein to produce fatty acid derivatives, and in some examplescan be expressed with additional enzymes to produce fatty acidderivatives having desired carbon chain characteristics. Exemplary fattyacid derivatives include for example, fatty acids, acyl-CoA, fattyaldehyde, short and long chain alcohols, hydrocarbons, fatty alcohols,and esters (e.g., waxes, fatty acid esters, or fatty esters).

As used herein, the term “fatty acid derivative enzymes” means allenzymes that may be expressed or overexpressed in the production offatty acid derivatives. These enzymes are collectively referred toherein as fatty acid derivative enzymes. These enzymes may be part ofthe fatty acid biosynthetic pathway. Non-limiting examples of fatty acidderivative enzymes include fatty acid synthases, thioesterases, acyl-CoAsynthases, acyl-CoA reductases, alcohol dehydrogenases, alcoholacyltransferases, fatty alcohol-forming acyl-CoA reductase, estersynthases, aldehyde biosynthetic polypeptides, and alkane biosyntheticpolypeptides. Fatty acid derivative enzymes convert a substrate into afatty acid derivative. In some examples, the substrate may be a fattyacid derivative which the fatty acid derivative enzyme converts into adifferent fatty acid derivative.

As used herein, the term “fatty alcohol forming peptides” means apeptide capable of catalyzing the conversion of acyl-CoA to fattyalcohol, including fatty alcohol forming acyl-CoA reductase (FAR, EC1.1.1.*), acyl-CoA reductase (EC 1.2.1.50), or alcohol dehydrogenase (EC1.1.1.1). Additionally, one of ordinary skill in the art will appreciatethat some fatty alcohol forming peptides will catalyze other reactionsas well. For example, some acyl-CoA reductase peptides will accept othersubstrates in addition to fatty acids. Such non-specific peptides are,therefore, also included. Nucleic acid sequences encoding fatty alcoholforming peptides are known in the art, and such peptides are publiclyavailable. Exemplary GenBank Accession Numbers are provided in FIG. 40.

As used herein, “fatty acid enzyme” means any enzyme involved in fattyacid biosynthesis. Fatty acid enzymes can be expressed or overexpressedin host cells to produce fatty acids. Non-limiting examples of fattyacid enzymes include fatty acid synthases and thioesterases.

As used herein, the term “fatty ester” means an ester. In a preferredembodiment, a fatty ester is any ester made from a fatty acid, forexample a fatty acid ester. In one embodiment, a fatty ester contains anA side (i.e., the carbon chain attached to the carboxylate oxygen) and aB side (i.e., the carbon chain comprising the parent carboxylate). In apreferred embodiment, when the fatty ester is derived from the fattyacid biosynthetic pathway, the A side is contributed by an alcohol, andthe B side is contributed by a fatty acid. Any alcohol can be used toform the A side of the fatty esters. For example, the alcohol can bederived from the fatty acid biosynthetic pathway. Alternatively, thealcohol can be produced through non-fatty acid biosynthetic pathways.Moreover, the alcohol can be provided exogenously. For example, thealcohol can be supplied in the fermentation broth in instances where thefatty ester is produced by an organism. Alternatively, a carboxylicacid, such as a fatty acid or acetic acid, can be supplied exogenouslyin instances where the fatty ester is produced by an organism that canalso produce alcohol.

The carbon chains comprising the A side or B side can be of any length.In one embodiment, the A side of the ester is at least about 1, 2, 3, 4,5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. The B side of theester is at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26carbons in length. The A side and/or the B side can be straight orbranched chain. The branched chains may have one or more points ofbranching. In addition, the branched chains may include cyclic branches.Furthermore, the A side and/or B side can be saturated or unsaturated.If unsaturated, the A side and/or B side can have one or more points ofunsaturation.

In one embodiment, the fatty ester is produced biosynthetically. In thisembodiment, first the fatty acid is “activated.” Non-limiting examplesof “activated” fatty acids are acyl-CoA, acyl-ACP, and acyl phosphate.Acyl-CoA can be a direct product of fatty acid biosynthesis ordegradation. In addition, acyl-CoA can be synthesized from a free fattyacid, a CoA, or an adenosine nucleotide triphosphate (ATP). An exampleof an enzyme which produces acyl-CoA is acyl-CoA synthase

After the fatty acid is activated, it can be readily transferred to arecipient nucleophile. Exemplary nucleophiles are alcohols, thiols, orphosphates.

In one embodiment, the fatty ester is a wax. The wax can be derived froma long chain alcohol and a long chain fatty acid. In another embodiment,the fatty ester can be derived from a fatty acyl-thioester and analcohol. In another embodiment, the fatty ester is a fatty acidthioester, for example fatty acyl Coenzyme A (CoA). In otherembodiments, the fatty ester is a fatty acyl panthothenate, an acylcarrier protein (ACP), or a fatty phosphate ester. Fatty esters havemany uses. For example, fatty esters can be used as a biofuel.

As used herein, “fraction of modern carbon” or “f_(M)” has the samemeaning as defined by National Institute of Standards and Technology(NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known asoxalic acids standards HOxI and HOxII, respectively. The fundamentaldefinition relates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI(referenced to AD 1950). This is roughly equivalent to decay-correctedpre-Industrial Revolution wood. For the current living biosphere (plantmaterial), f_(M) is approximately 1.1.

Calculations of “homology” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence that isaligned for comparison purposes is at least about 30%, preferably atleast about 40%, more preferably at least about 50%, even morepreferably at least about 60%, and even more preferably at least about70%, at least about 80%, at least about 90%, or about 100% of the lengthof the reference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein, amino acid or nucleic acid “identity” is equivalent toamino acid or nucleic acid “homology”). The percent identity between thetwo sequences is a function of the number of identical positions sharedby the sequences, taking into account the number of gaps and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent homology between twoamino acid sequences is determined using the Needleman and Wunsch(1970), J. Mol. Biol. 48:444 453, algorithm that has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent homology between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used if thepractitioner is uncertain about which parameters should be applied todetermine if a molecule is within a homology limitation of the claims)are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extendpenalty of 4, and a frameshift gap penalty of 5.

As used herein, a “host cell” is a cell used to produce a productdescribed herein (e.g., an aldehyde or alkane described herein). A hostcell can be modified to express or overexpress selected genes or to haveattenuated expression of selected genes. Non-limiting examples of hostcells include plant, animal, human, bacteria, yeast, or filamentousfungi cells.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueousand nonaqueous methods are described in that reference and either methodcan be used. Specific hybridization conditions referred to herein are asfollows: 1) low stringency hybridization conditions in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); 2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringencyhybridization conditions in 6×SSC at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very highstringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Veryhigh stringency conditions (4) are the preferred conditions unlessotherwise specified.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively, that are present in the natural source of the nucleicacid. Moreover, an “isolated nucleic acid” includes nucleic acidfragments, such as fragments that are not naturally occurring. The term“isolated” is also used herein to refer to polypeptides, which areisolated from other cellular proteins, and encompasses both purifiedendogenous polypeptides and recombinant polypeptides. The term“isolated” as used herein also refers to a nucleic acid or polypeptidethat is substantially free of cellular material, viral material, orculture medium when produced by recombinant DNA techniques. The term“isolated” as used herein also refers to a nucleic acid or polypeptidethat is substantially free of chemical precursors or other chemicalswhen chemically synthesized.

As used herein, the “level of expression of a gene in a cell” refers tothe level of mRNA, pre-mRNA nascent transcript(s), transcript processingintermediates, mature mRNA(s), and/or degradation products encoded bythe gene in the cell.

As used herein, the term “microorganism” means prokaryotic andeukaryotic microbial species from the domains Archaea, Bacteria andEucarya, the latter including yeast and filamentous fungi, protozoa,algae, or higher Protista. The term “microbial cell”, as used herein,means a cell from a microorganism.

As used herein, the term “nucleic acid” refers to polynucleotides, suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term also includes analogs of either RNA or DNA made fromnucleotide analogs, and, as applicable to the embodiment beingdescribed, single (sense or antisense) and double-strandedpolynucleotides, ESTs, chromosomes, cDNAs, mRNAs, and rRNAs.

As used herein, the term “operably linked” means that a selectednucleotide sequence (e.g., encoding a polypeptide described herein) isin proximity with a promoter to allow the promoter to regulateexpression of the selected nucleotide sequence. In addition, thepromoter is located upstream of the selected nucleotide sequence interms of the direction of transcription and translation. By “operablylinked” is meant that a nucleotide sequence and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequence(s).

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “overexpress” means to express or cause to be expresseda nucleic acid, polypeptide, or hydrocarbon in a cell at a greaterconcentration than is normally expressed in a corresponding wild-typecell. For example, a polypeptide can be “overexpressed” in a recombinanthost cell when the polypeptide is present in a greater concentration inthe recombinant host cell compared to its concentration in anon-recombinant host cell of the same species.

As used herein, “partition coefficient” or “P,” is defined as theequilibrium concentration of a compound in an organic phase divided bythe concentration at equilibrium in an aqueous phase (e.g., fermentationbroth). In one embodiment of a bi-phasic system described herein, theorganic phase is formed by the aldehyde or alkane during the productionprocess. However, in some examples, an organic phase can be provided,such as by providing a layer of octane, to facilitate productseparation. When describing a two phase system, the partitioncharacteristics of a compound can be described as log P. For example, acompound with a log P of 1 would partition 10:1 to the organic phase. Acompound with a log P of −1 would partition 1:10 to the organic phase.By choosing an appropriate fermentation broth and organic phase, analdehyde or alkane with a high log P value can separate into the organicphase even at very low concentrations in the fermentation vessel.

As used herein, the term “purify,” “purified,” or “purification” meansthe removal or isolation of a molecule from its environment by, forexample, isolation or separation. “Substantially purified” molecules areat least about 60% free, preferably at least about 75% free, and morepreferably at least about 90% free from other components with which theyare associated. As used herein, these terms also refer to the removal ofcontaminants from a sample. For example, the removal of contaminants canresult in an increase in the percentage of aldehydes or alkanes in asample. For example, when aldehydes or alkanes are produced in a hostcell, the aldehydes or alkanes can be purified by the removal of hostcell proteins. After purification, the percentage of aldehydes oralkanes in the sample is increased.

The terms “purify,” “purified,” and “purification” do not requireabsolute purity. They are relative terms. Thus, for example, whenaldehydes or alkanes are produced in host cells, a purified aldehyde orpurified alkane is one that is substantially separated from othercellular components (e.g., nucleic acids, polypeptides, lipids,carbohydrates, or other hydrocarbons). In another example, a purifiedaldehyde or purified alkane preparation is one in which the aldehyde oralkane is substantially free from contaminants, such as those that mightbe present following fermentation. In some embodiments, an aldehyde oran alkane is purified when at least about 50% by weight of a sample iscomposed of the aldehyde or alkane. In other embodiments, an aldehyde oran alkane is purified when at least about 60%, 70%, 80%, 85%, 90%, 92%,95%, 98%, or 99% or more by weight of a sample is composed of thealdehyde or alkane.

As used herein, the term “recombinant polypeptide” refers to apolypeptide that is produced by recombinant DNA techniques, whereingenerally DNA encoding the expressed polypeptide or RNA is inserted intoa suitable expression vector and that is in turn used to transform ahost cell to produce the polypeptide or RNA.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used to refer to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleotide sequence such that the first and second amino acid ornucleotide sequences have similar activities.

As used herein, the term “synthase” means an enzyme which catalyzes asynthesis process. As used herein, the term synthase includes synthases,synthetases, and ligases.

As used herein, the term “transfection” means the introduction of anucleic acid (e.g., via an expression vector) into a recipient cell bynucleic acid-mediated gene transfer.

As used herein, “transformation” refers to a process in which a cell'sgenotype is changed as a result of the cellular uptake of exogenousnucleic acid. This may result in the transformed cell expressing arecombinant form of an RNA or polypeptide. In the case of antisenseexpression from the transferred gene, the expression of anaturally-occurring form of the polypeptide is disrupted.

As used herein, a “transport protein” is a polypeptide that facilitatesthe movement of one or more compounds in and/or out of a cellularorganelle and/or a cell.

As used herein, a “variant” of polypeptide X refers to a polypeptidehaving the amino acid sequence of polypeptide X in which one or moreamino acid residues is altered. The variant may have conservativechanges or nonconservative changes. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted without affectingbiological activity may be found using computer programs well known inthe art, for example, LASERGENE software (DNASTAR).

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to that of agene or the coding sequence thereof. This definition may also include,for example, “allelic,” “splice,” “species,” or “polymorphic” variants.A splice variant may have significant identity to a referencepolynucleotide, but will generally have a greater or fewer number ofpolynucleotides due to alternative splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or an absence of domains. Species variants arepolynucleotide sequences that vary from one species to another. Theresulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of useful vector is an episome (i.e., a nucleic acidcapable of extra-chromosomal replication). Useful vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids,” whichrefer generally to circular double stranded DNA loops that, in theirvector form, are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably, as theplasmid is the most commonly used form of vector. However, also includedare such other forms of expression vectors that serve equivalentfunctions and that become known in the art subsequently hereto.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a GC/MS trace of hydrocarbons produced by Prochlorococcusmarinus CCMP1986 cells. FIG. 1B is a mass fragmentation pattern of thepeak at 7.55 min of FIG. 1A.

FIG. 2A is a GC/MS trace of hydrocarbons produced by Nostoc punctiformePCC73102 cells. FIG. 2B is a mass fragmentation pattern of the peak at8.73 min of FIG. 2A.

FIG. 3A is a GC/MS trace of hydrocarbons produced by Gloeobaceterviolaceus ATCC29082 cells. FIG. 3B is a mass fragmentation pattern ofthe peak at 8.72 min of FIG. 3A.

FIG. 4A is a GC/MS trace of hydrocarbons produced by Synechocystic sp.PCC6803 cells. FIG. 4B is a mass fragmentation pattern of the peak at7.36 min of FIG. 4A.

FIG. 5A is a GC/MS trace of hydrocarbons produced by Synechocystis sp.PCC6803 wild type cells. FIG. 5B is a GC/MS trace of hydrocarbonsproduced by Synechocystis sp. PCC6803 cells with a deletion of thesll0208 and sll0209 genes.

FIG. 6A is a GC/MS trace of hydrocarbons produced by E. coli MG1655 wildtype cells. FIG. 6B is a GC/MS trace of hydrocarbons produced by E. coliMG1655 cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65).

FIG. 7 is a GC/MS trace of hydrocarbons produced by E. coli cellsexpressing Cyanothece sp. ATCC51142 cce_1430 (YP_001802846) (SEQ IDNO:69).

FIG. 8A is a GC/MS trace of hydrocarbons produced by E. coli cellsexpressing Synechococcus elongatus PCC7942 YP_400611 (Synpcc7942_1594)(SEQ ID NO:65) and Synechococcus elongatus PCC7942 YP_400610(Synpcc7942_1593) (SEQ ID NO:1). FIG. 8B depicts mass fragmentationpatterns of the peak at 6.98 min of FIG. 8A and of pentadecane. FIG. 8Cdepicts mass fragmentation patterns of the peak at 8.12 min of FIG. 8Aand of 8-heptadecene.

FIG. 9 is a GC/MS trace of hydrocarbons produced by E. coli MG1655 cellsexpressing Synechococcus elongatus PCC7942 YP_400611 (Synpcc7942_1594)(SEQ ID NO:65) and Nostoc punctiforme PCC73102 Npun02004178(ZP_00108838) (SEQ ID NO:5).

FIG. 10 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and Synechocystis sp. PCC6803 sll0208(NP_442147) (SEQ ID NO:3).

FIG. 11 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and Nostoc sp. PCC7210 alr5283(NP_489323) (SEQ ID NO:7).

FIG. 12 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized Acaryochlorismarina MBIC11017 AM1_4041 (YP_001518340) (SEQ ID NO:46).

FIG. 13 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized Thermosynechococcuselongatus BP-1 tll1313 (NP_682103) (SEQ ID NO:47).

FIG. 14 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized Synechococcus sp.JA-3-3Ab CYA_0415 (YP_473897) (SEQ ID NO:48).

FIG. 15 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and Gloeobacter violaceus PCC7421gll3146 (NP_926092) (SEQ ID NO:15).

FIG. 16 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized Prochlorococcusmarinus MIT9313 PMT1231 (NP_895059) (SEQ ID NO:49).

FIG. 17 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and Prochlorococcus marinus CCMP1986PMM0532 (NP_892650) (SEQ ID NO:19).

FIG. 18 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized Prochlorococcusmariunus NATL2A PMN2A_1863 (YP_293054) (SEQ ID NO:51).

FIG. 19 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized Synechococcus sp.RS9917 RS9917_09941 (ZP_01079772) (SEQ ID NO:52).

FIG. 20 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized Synechococcus sp.RS9917 RS9917_12945 (ZP_01080370) (SEQ ID NO:53).

FIG. 21 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and Cyanothece sp. ATCC51142 cce_0778(YP_001802195) (SEQ ID NO:27).

FIG. 22 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and Cyanothece sp. PCC7425Cyan7425_0398 (YP_002481151) (SEQ ID NO:29).

FIG. 23 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and Cyanothece sp. PCC7425Cyan7425_2986 (YP_002483683) (SEQ ID NO:31).

FIG. 24A is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Prochlorococcus marinus CCMP1986 PMM0533 (NP_892651)(SEQ ID NO:71). FIG. 24B is a GC/MS trace of hydrocarbons produced by E.coli MG1655 cells expressing Prochlorococcus marinus CCMP1986 PMM0533(NP_892651) (SEQ ID NO:71) and Prochlorococcus mariunus CCMP1986 PMM0532(NP_892650) (SEQ ID NO:19).

FIG. 25A is a GC/MS trace of hydrocarbons produced by E. coli MG1655ΔfadE lacZ::P_(trc) ′tesA-fadD cells. FIG. 25B is a GC/MS trace ofhydrocarbons produced by E. coli MG1655 ΔfadE lacZ::P_(trc) ′tesA-fadDcells expressing Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:65) and Acaryochloris marina MBIC11017AM1_4041 (YP_001518340) (SEQ ID NO:9).

FIG. 26A is a GC/MS trace of hydrocarbons produced by E. coli MG1655ΔfadE lacZ::P_(trc) ′tesA-fadD cells expressing Synechocystis sp.PCC6803 sll0209 (NP_442146) (SEQ ID NO:67). FIG. 26B is a GC/MS trace ofhydrocarbons produced by E. coli MG1655 ΔfadE lacZ::P_(trc) ′tesA-fadDcells expressing Synechocystis sp. PCC6803 sll0209 (NP_442146) (SEQ IDNO:67) and Synechocystis sp. PCC6803 sll0208 (NP_442147) (SEQ ID NO:3).

FIG. 27A is a GC/MS trace of hydrocarbons produced by E. coli MG1655ΔfadD lacZ::P_(trc)-′tesA cells expressing M. smegmatis strain MC2 155MSMEG_5739 (YP_889972) (SEQ ID NO:85). FIG. 27B is a GC/MS trace ofhydrocarbons produced by E. coli MG1655 ΔfadD lacZ::P_(trc)-′tesA cellsexpressing M. smegmatis strain MC2 155 MSMEG_5739 (YP_889972) (SEQ IDNO:85) and Nostoc punctiforme PCC73102 Npun02004178 (ZP_00108838) (SEQID NO:5).

FIG. 28 is a graphic representation of hydrocarbons produced by E. coliMG1655 ΔfadD lacZ::P_(trc)-′tesA cells expressing M. smegmatis strainMC2 155 MSMEG_5739 (YP_889972) (SEQ ID NO:85) either alone or incombination with Nostoc sp. PCC7120 alr5283 (SEQ ID NO:7), Nostocpunctiforme PCC73102 Npun02004178 (SEQ ID NO:5), P. mariunus CCMP1986PMM0532 (SEQ ID NO:19), G. violaceus PCC7421 gll3146 (SEQ ID NO:15),Synechococcus sp. RS9917_09941 (SEQ ID NO:23), Synechococcus sp.RS9917_12945 (SEQ ID NO:25), or A. marina MBIC11017 AM1_4041 (SEQ IDNO:9).

FIG. 29A is a representation of the three-dimensional structure of aclass I ribonuclease reductase subunit β protein, RNRβ. FIG. 29B is arepresentation of the three-dimensional structure of Prochlorococcusmarinus MIT9313 PMT1231 (NP_895059) (SEQ ID NO:17). FIG. 29C is arepresentation of the three-dimensional structure of the active site ofProchlorococcus marinus MIT9313 PMT1231 (NP_895059) (SEQ ID NO:17).

FIG. 30A is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Nostoc punctiforme PCC73102 Npun02004178 (ZP_00108838)(SEQ ID NO:5). FIG. 30B is a GC/MS trace of hydrocarbons produced by E.coli MG1655 cells expressing Nostoc punctiforme PCC73102 Npun02004178(ZP_00108838) Y123F variant. FIG. 30C is a GC/MS trace of hydrocarbonsproduced by E. coli MG1655 cells expressing Nostoc punctiforme PCC73102Npun02004178 (ZP_00108838) Y126F variant.

FIG. 31 depicts GC/MS traces of hydrocarbons produced in vitro usingNostoc punctiforme PCC73102 Npun02004178 (ZP_00108838) (SEQ ID NO:6) andoctadecanal (A); Npun02004178 (ZP_00108838) (SEQ ID NO:6), octadecanal,spinach ferredoxin reductase, and NADPH (B); octadecanal, spinachferredoxin, spinach ferredoxin reductase, and NADPH (C); or Npun02004178(ZP_00108838) (SEQ ID NO:6), spinach ferredoxin, and spinach ferredoxin(D).

FIG. 32 depicts GC/MS traces of hydrocarbons produced in vitro usingNostoc punctiforme PCC73102 Npun02004178 (ZP_00108838) (SEQ ID NO:6),NADPH, octadecanal, and either (A) spinach ferredoxin and spinachferredoxin reductase; (B) N. punctiforme PCC73102 Npun02003626(ZP_00109192) (SEQ ID NO:88) and N. punctiforme PCC73102 Npun02001001(ZP_00111633) (SEQ ID NO:90); (C) Npun02003626 (ZP_00109192) (SEQ IDNO:88) and N. punctiforme PCC73102 Npun02003530 (ZP_00109422) (SEQ IDNO:92); or (D) Npun02003626 (ZP_00109192) (SEQ ID NO:88) and N.punctiforme PCC73102 Npun02003123 (ZP_00109501) (SEQ ID NO:94).

FIG. 33A is a GC/MS trace of hydrocarbons produced in vitro usingoctadecanoyl-CoA, Synechococcus elongatus PCC7942 YP_400611(Synpcc7942_1594) (SEQ ID NO:66), NADH, and Mg²⁺. FIG. 33B is a GC/MStrace of hydrocarbons produced in vitro using octadecanoyl-CoA,Synechococcus elongatus PCC7942 YP_400611 (Synpcc7942_1594) (SEQ IDNO:66), NADPH, and Mg²⁺. FIG. 33C is a GC/MS trace of hydrocarbonsproduced in vitro using octadecanoyl-CoA, Synechococcus elongatusPCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:66) and NADPH.

FIG. 34A is a GC/MS trace of hydrocarbons produced in vitro usingoctadecanoyl-CoA, labeled NADPH, Synechococcus elongatus PCC7942YP_400611 (Synpcc7942_1594) (SEQ ID NO:66), and unlabeled NADPH. FIG.34B is a GC/MS trace of hydrocarbons produced in vitro usingoctadecanoyl-CoA, labeled NADPH, Synechococcus elongatus PCC7942YP_400611 (Synpcc7942_1594) (SEQ ID NO:66), and S-(4-²H)NADPH. FIG. 34Cis a GC/MS trace of hydrocarbons produced in vitro usingoctadecanoyl-CoA, labeled NADPH, Synechococcus elongatus PCC7942YP_400611 (Synpcc7942_1594) (SEQ ID NO:66), and R-(4-²H)NADPH.

FIG. 35 is a GC/MS trace of hydrocarbons in the cell-free supernatantproduced by E. coli MG1655 ΔfadE cells in Che-9 media expressingSynechococcus elongatus PCC7942 YP_400611 (Synpcc7942_1594) (SEQ IDNO:65).

FIG. 36 is a GC/MS trace of hydrocarbons in the cell-free supernatantproduced by E. coli MG1655 ΔfadE cells in Che-9 media expressingSynechococcus elongatus PCC7942 YP_400611 (Synpcc7942_1594) (SEQ IDNO:65) and Nostoc punctiforme PCC73102 Npun02004178 (ZP_00108838) (SEQID NO:5).

FIG. 37 is a GC/MS trace of hydrocarbons produced by E. coli MG1655cells expressing Nostoc sp. PCC7120 alr5283 (NP_489323) (SEQ ID NO:7)and Nostoc sp. PCC7120 alr5284 (NP_489324) (SEQ ID NO:81).

FIG. 38A-38D is a list of examples of homologs of Synechococcuselongatus PCC7942 YP_400610 (Synpcc7942_1593) (SEQ ID NO:1) from ametagenomic database.

FIG. 39A-39D is a list of examples of homologs of Synechococcuselongatus PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) from ametagenomic database.

FIG. 40A-40T is a table identifying various genes that can be expressed,overexpressed, or attenuated to increase production of particularsubstrates.

DETAILED DESCRIPTION

The invention provides compositions and methods of producing aldehydes,fatty alcohols, and hydrocarbons (such as alkanes, alkenes, and alkynes)from substrates, for example, an acyl-ACP, a fatty acid, an acyl-CoA, afatty aldehyde, or a fatty alcohol substrate (e.g., as described inPCT/US08/058788, specifically incorporated by reference herein). Suchaldehydes, alkanes, and alkenes are useful as biofuels (e.g.,substitutes for gasoline, diesel, jet fuel, etc.), specialty chemicals(e.g., lubricants, fuel additive, etc.), or feedstock for furtherchemical conversion (e.g., fuels, polymers, plastics, textiles,solvents, adhesives, etc.). The invention is based, in part, on theidentification of genes that are involved in aldehyde, alkane, andalkene biosynthesis.

Such alkane and alkene biosynthetic genes include, for example,Synechococcus elongatus PCC7942 Synpcc7942_1593 (SEQ ID NO:1),Synechocystis sp. PCC6803 sll0208 (SEQ ID NO:3), Nostoc punctiforme PCC73102 Npun02004178 (SEQ ID NO:5), Nostoc sp. PCC 7120 alr5283 (SEQ IDNO:7), Acaryochloris marina MBIC11017 AM1_4041 (SEQ ID NO:9),Thermosynechococcus elongatus BP-1 tll1313 (SEQ ID NO:11), Synechococcussp. JA-3-3A CYA_0415 (SEQ ID NO:13), Gloeobacter violaceus PCC 7421gll3146 (SEQ ID NO:15), Prochlorococcus marinus MIT9313 PM123 (SEQ IDNO:17), Prochlorococcus marinus subsp. pastoris str. CCMP1986 PMM0532(SEQ ID NO:19), Prochlorococcus marinus str. NATL2A PMN2A_1863 (SEQ IDNO:21), Synechococcus sp. RS9917 RS9917_09941 (SEQ ID NO:23),Synechococcus sp. RS9917 RS9917_12945 (SEQ ID NO:25), Cyanothece sp.ATCC51142 cce_0778 (SEQ ID NO:27), Cyanothece sp. PCC7245Cyan7425DRAFT_1220 (SEQ ID NO:29), Cyanothece sp. PCC7245 cce_0778 (SEQID NO:31), Anabaena variabilis ATCC29413 YP_323043 (Ava_2533) (SEQ IDNO:33), and Synechococcus elongatus PCC6301 YP_170760 (syc0050_d) (SEQID NO:35). Other alkane and alkene biosynthetic genes are listed inTable 1 and FIG. 38.

Aldehyde biosynthetic genes include, for example, Synechococcuselongatus PCC7942 Synpcc7942_1594 (SEQ ID NO:65), Synechocystis sp.PCC6803 sll0209 (SEQ ID NO:67), Cyanothece sp. ATCC51142 cce_1430 (SEQID NO:69), Prochlorococcus marinus subsp. pastoris str. CCMP1986 PMM0533(SEQ ID NO:71), Gloeobacter violaceus PCC7421 NP_96091 (gll3145) (SEQ IDNO:73), Nostoc punctiforme PCC73102 ZP_00108837 (Npun02004176) (SEQ IDNO:75), Anabaena variabilis ATCC29413 YP_323044 (Ava_2534) (SEQ IDNO:77), Synechococcus elongatus PCC6301 YP_170761 (syc0051_d) (SEQ IDNO:79), and Nostoc sp. PCC 7120 alr5284 (SEQ ID NO:81). Other aldehydebiosynthetic genes are listed in Table 1 and FIG. 39.

Using the methods described herein, aldehydes, fatty alcohols, alkanes,and alkenes can be prepared using one or more aldehyde, alkane, and/oralkene biosynthetic genes or polypeptides described herein, or variantsthereof, utilizing host cells or cell-free methods.

TABLE 1 Aldehyde and alkane biosynthetic gene homologs in cyanobacterialgenomes Alkane Biosynth. Gene Aldehyde Biosynth. Gene Cyanobacteriumaccession number % ID accession number % ID Synechococcus elongatus PCC7942 YP_400610 100 YP_400611 100 Synechococcus elongatus PCC 6301YP_170760 100 YP_170761 100 Microcoleus chthonoplastes PCC 7420 EDX7501977 EDX74978 70 Arthrospira maxima CS-328 EDZ94963 78 EDZ94968 68 Lyngbyasp. PCC 8106 ZP_01619575 77 ZP_01619574 69 Nodularia spumigena CCY9414ZP_01628096 77 ZP_01628095 70 Trichodesmium erythraeum IMS101 YP_72197976 YP_721978 69 Microcystis aeruginosa NIES-843 YP_001660323 75YP_001660322 68 Microcystis aeruginosa PCC 7806 CAO90780 74 CAO90781 67Nostoc sp. PCC 7120 NP_489323 74 NP_489324 72 Nostoc azollae 0708EEG05692 73 EEG05693 70 Anabaena variabilis ATCC 29413 YP_323043 74YP_323044 73 Crocosphaera watsonii WH 8501 ZP_00514700 74 ZP_00516920 67Synechocystis sp. PCC 6803 NP_442147 72 NP_442146 68 Synechococcus sp.PCC 7335 EDX86803 73 EDX87870 67 Cyanothece sp. ATCC 51142 YP_00180219573 YP_001802846 67 Cyanothece sp. CCY0110 ZP_01728578 72 ZP_01728620 68Nostoc punctiforme PCC 73102 ZP_00108838 72 ZP_00108837 71 Acaryochlorismarina MBIC11017 YP_001518340 71 YP_001518341 66 Cyanothece sp. PCC 7425YP_002481151 71 YP_002481152 70 Cyanothece sp. PCC 8801 ZP_02941459 70ZP_02942716 69 Thermosynechococcus elongatus BP-1 NP_682103 70 NP_68210270 Synechococcus sp. JA-2-3B′a(2-13) YP_478639 68 YP_478638 63Synechococcus sp. RCC307 YP_001227842 67 YP_001227841 64 Synechococcussp. WH 7803 YP_001224377 68 YP_001224378 65 Synechococcus sp. WH 8102NP_897829 70 NP_897828 65 Synechococcus sp. WH 7805 ZP_01123214 68ZP_01123215 65 uncultured marine type-A Synechococcus GOM 3O12 ABD9637670 ABD96375 65 Synechococcus sp. JA-3-3Ab YP_473897 68 YP_473896 62uncultured marine type-A Synechococcus GOM 3O6 ABD96328 70 ABD96327 65uncultured marine type-A Synechococcus GOM 3M9 ABD96275 68 ABD96274 65Synechococcus sp. CC9311 YP_731193 63 YP_731192 63 uncultured marinetype-A Synechococcus 5B2 ABB92250 69 ABB92249 64 Synechococcus sp. WH5701 ZP_01085338 66 ZP_01085337 67 Gloeobacter violaceus PCC 7421NP_926092 63 NP_926091 67 Synechococcus sp. RS9916 ZP_01472594 69ZP_01472595 66 Synechococcus sp. RS9917 ZP_01079772 68 ZP_01079773 65Synechococcus sp. CC9605 YP_381055 66 YP_381056 66 Cyanobium sp. PCC7001 EDY39806 64 EDY38361 64 Prochlorococcus marinus str. MIT 9303YP_001016795 63 YP_001016797 66 Prochlorococcus marinus str. MIT9313NP_895059 63 NP_895058 65 Synechococcus sp. CC9902 YP_377637 66YP_377636 65Aldehyde, Alkane, and Alkene Biosynthetic Genes and Variants

The methods and compositions described herein include, for example,alkane or alkene biosynthetic genes having the nucleotide sequence ofSEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,or 35, as well as polynucleotide variants thereof. In some instances,the alkane or alkene biosynthetic gene encodes one or more of the aminoacid motifs described herein. For example, the alkane or alkenebiosynthetic gene can encode a polypeptide comprising SEQ ID NO:37, 38,39, 41, 42, 43, or 44. The alkane or alkene biosynthetic gene can alsoinclude a polypeptide comprising SEQ ID NO:40 and also any one of SEQ IDNO:37, 38, or 39.

The methods and compositions described herein also include, for example,aldehyde biosynthetic genes having the nucleotide sequence of SEQ IDNO:65, 67, 69, 71, 73, 75, 77, 79, or 81, as well as polynucleotidevariants thereof. In some instances, the aldehyde biosynthetic geneencodes one or more of the amino acid motifs described herein. Forexample, the aldehyde biosynthetic gene can encode a polypeptidecomprising SEQ ID NO:54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64.

The variants can be naturally occurring or created in vitro. Inparticular, such variants can be created using genetic engineeringtechniques, such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives can be created using chemical synthesis or modificationprocedures.

Methods of making variants are well known in the art. These includeprocedures in which nucleic acid sequences obtained from naturalisolates are modified to generate nucleic acids that encode polypeptideshaving characteristics that enhance their value in industrial orlaboratory applications. In such procedures, a large number of variantsequences having one or more nucleotide differences with respect to thesequence obtained from the natural isolate are generated andcharacterized. Typically, these nucleotide differences result in aminoacid changes with respect to the polypeptides encoded by the nucleicacids from the natural isolates.

For example, variants can be created using error prone PCR (see, e.g.,Leung et al., Technique 1:11-15, 1989; and Caldwell et al., PCR MethodsApplic. 2:28-33, 1992). In error prone PCR, PCR is performed underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. Briefly, in such procedures, nucleic acids to bemutagenized (e.g., an aldehyde or alkane biosynthetic polynucleotidesequence), are mixed with PCR primers, reaction buffer, MgCl₂, MnCl₂,Taq polymerase, and an appropriate concentration of dNTPs for achievinga high rate of point mutation along the entire length of the PCRproduct. For example, the reaction can be performed using 20 fmoles ofnucleic acid to be mutagenized (e.g., an aldehyde or alkane biosyntheticpolynucleotide sequence), 30 pmole of each PCR primer, a reaction buffercomprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), and 0.01% gelatin, 7 mMMgCl₂, 0.5 mM MnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mMdATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performed for 30 cycles of94° C. for 1 min, 45° C. for 1 min, and 72° C. for 1 min. However, itwill be appreciated that these parameters can be varied as appropriate.The mutagenized nucleic acids are then cloned into an appropriate vectorand the activities of the polypeptides encoded by the mutagenizednucleic acids are evaluated.

Variants can also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olsonet al., Science 241:53-57, 1988. Briefly, in such procedures a pluralityof double stranded oligonucleotides bearing one or more mutations to beintroduced into the cloned DNA are synthesized and inserted into thecloned DNA to be mutagenized (e.g., an aldehyde or alkane biosyntheticpolynucleotide sequence). Clones containing the mutagenized DNA arerecovered, and the activities of the polypeptides they encode areassessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, for example, U.S.Pat. No. 5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different, but highly related, DNA sequence invitro as a result of random fragmentation of the DNA molecule based onsequence homology. This is followed by fixation of the crossover byprimer extension in a PCR reaction. Sexual PCR mutagenesis is describedin, for example, Stemmer, PNAS, USA 91:10747-10751, 1994.

Variants can also be created by in vivo mutagenesis. In someembodiments, random mutations in a nucleic acid sequence are generatedby propagating the sequence in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type strain. Propagating a DNA sequence (e.g., analdehyde or alkane biosynthetic polynucleotide sequence) in one of thesestrains will eventually generate random mutations within the DNA.Mutator strains suitable for use for in vivo mutagenesis are describedin, for example, PCT Publication No. WO 91/16427.

Variants can also be generated using cassette mutagenesis. In cassettemutagenesis, a small region of a double stranded DNA molecule isreplaced with a synthetic oligonucleotide “cassette” that differs fromthe native sequence. The oligonucleotide often contains a completelyand/or partially randomized native sequence.

Recursive ensemble mutagenesis can also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(i.e., protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in, for example, Arkin et al., PNAS, USA89:7811-7815, 1992.

In some embodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is describedin, for example, Delegrave et al., Biotech. Res. 11:1548-1552, 1993.Random and site-directed mutagenesis are described in, for example,Arnold, Curr. Opin. Biotech. 4:450-455, 1993.

In some embodiments, variants are created using shuffling procedureswherein portions of a plurality of nucleic acids that encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences that encode chimeric polypeptides as described in, forexample, U.S. Pat. Nos. 5,965,408 and 5,939,250.

Polynucleotide variants also include nucleic acid analogs. Nucleic acidanalogs can be modified at the base moiety, sugar moiety, or phosphatebackbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine and 5-methyl-2′-deoxycytidine or5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered, morpholino ring, orpeptide nucleic acids, in which the deoxyphosphate backbone is replacedby a pseudopeptide backbone and the four bases are retained. (See, e.g.,Summerton et al., Antisense Nucleic Acid Drug Dev. (1997) 7:187-195; andHyrup et al., Bioorgan. Med. Chem. (1996) 4:5-23.) In addition, thedeoxyphosphate backbone can be replaced with, for example, aphosphorothioate or phosphorodithioate backbone, a phosphoroamidite, oran alkyl phosphotriester backbone.

The aldehyde and alkane biosynthetic polypeptides Synpcc7942_1594 (SEQID N0:66) and Synpcc7942_1593 (SEQ ID NO:2) have homologs in othercyanobacteria (nonlimiting examples are depicted in Table 1). Thus, anypolynucleotide sequence encoding a homolog listed in Table 1, or avariant thereof, can be used as an aldehyde or alkane biosyntheticpolynucleotide in the methods described herein. Each cyanobacteriumlisted in Table 1 has copies of both genes. The level of sequenceidentity of the gene products ranges from 61% to 73% for Synpcc7942_1594(SEQ ID N0:66) and from 43% to 78% for Synpcc7942_1593 (SEQ ID N0:2).

Further homologs of the aldehyde biosynthetic polypeptideSynpcc7942_1594 (SEQ ID N0:66) are listed in FIG. 39, and anypolynucleotide sequence encoding a homolog listed in FIG. 39, or avariant thereof, can be used as an aldehyde biosynthetic polynucleotidein the methods described herein. Further homologs of the alkanebiosynthetic polypeptide Synpcc7942_1593 (SEQ ID NO:2) are listed inFIG. 38, and any polynucleotide sequence encoding a homolog listed inFIG. 38, or a variant thereof, can be used as an alkane biosyntheticpolynucleotide in the methods described herein.

In certain instances, an aldehyde, alkane, and/or alkene biosyntheticgene is codon optimized for expression in a particular host cell. Forexample, for expression in E. coli, one or more codons can be optimizedas described in, e.g., Grosjean et al., Gene 18:199-209 (1982).

Aldehyde, Alkane, and Alkene Biosynthetic Polypeptides and Variants

The methods and compositions described herein also include alkane oralkene biosynthetic polypeptides having the amino acid sequence of SEQID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or36, as well as polypeptide variants thereof. In some instances, analkane or alkene biosynthetic polypeptide is one that includes one ormore of the amino acid motifs described herein. For example, the alkaneor alkene biosynthetic polypeptide can include the amino acid sequenceof SEQ ID NO:37, 38, 39, 41, 42, 43, or 44. The alkane or alkenebiosynthetic polypeptide can also include the amino acid sequence of SEQID NO:40 and also any one of SEQ ID NO:37, 38, or 39.

The methods and compositions described herein also include aldehydebiosynthetic polypeptides having the amino acid sequence of SEQ IDNO:66, 68, 70, 72, 74, 76, 78, 80, or 82, as well as polypeptidevariants thereof. In some instances, an aldehyde biosyntheticpolypeptide is one that includes one or more of the amino acid motifsdescribed herein. For example, the aldehyde biosynthetic polypeptide caninclude the amino acid sequence of SEQ ID NO:54, 55, 56, 57, 58, 59, 60,61, 62, 63, or 64.

Aldehyde, alkane, and alkene biosynthetic polypeptide variants can bevariants in which one or more amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue). Such substituted amino acid residue may or may notbe one encoded by the genetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of similar characteristics.Typical conservative substitutions are the following replacements:replacement of an aliphatic amino acid, such as alanine, valine,leucine, and isoleucine, with another aliphatic amino acid; replacementof a serine with a threonine or vice versa; replacement of an acidicresidue, such as aspartic acid and glutamic acid, with another acidicresidue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue.

Other polypeptide variants are those in which one or more amino acidresidues include a substituent group. Still other polypeptide variantsare those in which the polypeptide is associated with another compound,such as a compound to increase the half-life of the polypeptide (e.g.,polyethylene glycol).

Additional polypeptide variants are those in which additional aminoacids are fused to the polypeptide, such as a leader sequence, asecretory sequence, a proprotein sequence, or a sequence whichfacilitates purification, enrichment, or stabilization of thepolypeptide.

In some instances, an alkane or alkene biosynthetic polypeptide variantretains the same biological function as a polypeptide having the aminoacid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, or 36 (e.g., retains alkane or alkene biosyntheticactivity) and has an amino acid sequence substantially identicalthereto.

In other instances, the alkane or alkene biosynthetic polypeptidevariants have at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or more than about 95% homology to the amino acid sequence of SEQID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or36. In another embodiment, the polypeptide variants include a fragmentcomprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or150 consecutive amino acids thereof.

In some instances, an aldehyde biosynthetic polypeptide variant retainsthe same biological function as a polypeptide having the amino acidsequence of SEQ ID NO:66, 68, 70, 72, 74, 76, 78, 80, or 82 (e.g.,retains aldehyde biosynthetic activity) and has an amino acid sequencesubstantially identical thereto.

In yet other instances, the aldehyde biosynthetic polypeptide variantshave at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, or morethan about 95% homology to the amino acid sequence of SEQ ID NO:66, 68,70, 72, 74, 76, 78, 80, or 82. In another embodiment, the polypeptidevariants include a fragment comprising at least about 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.

The polypeptide variants or fragments thereof can be obtained byisolating nucleic acids encoding them using techniques described hereinor by expressing synthetic nucleic acids encoding them. Alternatively,polypeptide variants or fragments thereof can be obtained throughbiochemical enrichment or purification procedures. The sequence ofpolypeptide variants or fragments can be determined by proteolyticdigestion, gel electrophoresis, and/or microsequencing. The sequence ofthe alkane or alkene biosynthetic polypeptide variants or fragments canthen be compared to the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 using any of theprograms described herein. The sequence of the aldehyde biosyntheticpolypeptide variants or fragments can be compared to the amino acidsequence of SEQ ID NO:66, 68, 70, 72, 74, 76, 78, 80, or 82 using any ofthe programs described herein.

The polypeptide variants and fragments thereof can be assayed foraldehyde-, fatty alcohol-, alkane-, and/or alkene-producing activityusing routine methods. For example, the polypeptide variants or fragmentcan be contacted with a substrate (e.g., a fatty acid derivativesubstrate or other substrate described herein) under conditions thatallow the polypeptide variant to function. A decrease in the level ofthe substrate or an increase in the level of an aldehyde, alkane, oralkene can be measured to determine aldehyde-, fatty alcohol-, alkane-,or alkene-producing activity, respectively.

Anti-Aldehyde, Anti-Fatty Alcohol, Anti-Alkane, and Anti-AlkeneBiosynthetic Polypeptide Antibodies

The aldehyde, fatty alcohol, alkane, and alkene biosyntheticpolypeptides described herein can also be used to produce antibodiesdirected against aldehyde, fatty alcohol, alkane, and alkenebiosynthetic polypeptides. Such antibodies can be used, for example, todetect the expression of an aldehyde, fatty alcohol, alkane, or alkenebiosynthetic polypeptide using methods known in the art. The antibodycan be, e.g., a polyclonal antibody; a monoclonal antibody or antigenbinding fragment thereof; a modified antibody such as a chimericantibody, reshaped antibody, humanized antibody, or fragment thereof(e.g., Fab′, Fab, F(ab)₂); or a biosynthetic antibody, e.g., a singlechain antibody, single domain antibody (DAB), Fv, single chain Fv(scFv), or the like.

Methods of making and using polyclonal and monoclonal antibodies aredescribed, e.g., in Harlow et al., Using Antibodies: A LaboratoryManual: Portable Protocol I. Cold Spring Harbor Laboratory (Dec. 1,1998). Methods for making modified antibodies and antibody fragments(e.g., chimeric antibodies, reshaped antibodies, humanized antibodies,or fragments thereof, e.g., Fab′, Fab, F(ab′)₂ fragments); orbiosynthetic antibodies (e.g., single chain antibodies, single domainantibodies (DABs), Fv, single chain Fv (scFv), and the like), are knownin the art and can be found, e.g., in Zola, Monoclonal Antibodies:Preparation and Use of Monoclonal Antibodies and Engineered AntibodyDerivatives, Springer Verlag (Dec. 15, 2000; 1st edition).

Substrates

The compositions and methods described herein can be used to producealdehydes, fatty alcohols, alkanes, and/or alkenes from an appropriatesubstrate. While not wishing to be bound by a particular theory, it isbelieved that the alkane or alkene biosynthetic polypeptides describedherein produce alkanes or alkenes from substrates via a decarbonylationmechanism. In some instances, the substrate is a fatty acid derivative,e.g., a fatty aldehyde, and an alkane having particular branchingpatterns and carbon chain length can be produced from a fatty acidderivative, e.g., a fatty aldehyde, having those particularcharacteristics. In other instances, the substrate is an unsaturatedfatty acid derivative, e.g., an unsaturated fatty aldehyde, and analkene having particular branching patterns and carbon chain length canbe produced from an unsaturated fatty acid derivative, e.g., anunsaturated fatty aldehyde, having those particular characteristics.

While not wishing to be bound by a particular theory, it is believedthat the aldehyde biosynthetic polypeptides described herein producealdehydes from substrates via a reduction mechanism. In certaininstances, the substrate is an acyl-ACP.

While not wishing to be bound by a particular theory, it is believedthat the fatty alcohols described herein are produced from substratesvia a reduction mechanism. In certain instances, the substrate is afatty aldehyde.

Accordingly, each step within a biosynthetic pathway that leads to theproduction of these substrates can be modified to produce or overproducethe substrate of interest. For example, known genes involved in thefatty acid biosynthetic pathway, the fatty aldehyde pathway, and thefatty alcohol pathway can be expressed, overexpressed, or attenuated inhost cells to produce a desired substrate (see, e.g., PCT/US08/058788,specifically incorporated by reference herein). Exemplary genes areprovided in FIG. 40.

Synthesis of Substrates

Fatty acid synthase (FAS) is a group of polypeptides that catalyze theinitiation and elongation of acyl chains (Marrakchi et al., BiochemicalSociety, 30:1050-1055, 2002). The acyl carrier protein (ACP) along withthe enzymes in the FAS pathway control the length, degree of saturation,and branching of the fatty acid derivatives produced. The fatty acidbiosynthetic pathway involves the precursors acetyl-CoA and malonyl-CoA.The steps in this pathway are catalyzed by enzymes of the fatty acidbiosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families (see,e.g., Heath et al., Prog. Lipid Res. 40(6):467-97 (2001)).

Host cells can be engineered to express fatty acid derivative substratesby recombinantly expressing or overexpressing acetyl-CoA and/ormalonyl-CoA synthase genes. For example, to increase acetyl-CoAproduction, one or more of the following genes can be expressed in ahost cell: pdh, panK, aceEF (encoding the E1p dehydrogenase componentand the E2p dihydrolipoamide acyltransferase component of the pyruvateand 2-oxoglutarate dehydrogenase complexes), fabH, fabD, fabG, acpP, andfabF. Exemplary GenBank accession numbers for these genes are: pdh(BAB34380, AAC73227, AAC73226), panK (also known as coaA, AAC76952),aceEF (AAC73227, AAC73226), fabH (AAC74175), fabD (AAC74176), fabG(AAC74177), acpP (AAC74178), fabF (AAC74179). Additionally, theexpression levels of fadE, gpsA, ldhA, pflb, adhE, pta, poxB, ackA,and/or ackB can be attenuated or knocked-out in an engineered host cellby transformation with conditionally replicative or non-replicativeplasmids containing null or deletion mutations of the correspondinggenes or by substituting promoter or enhancer sequences. ExemplaryGenBank accession numbers for these genes are: fadE (AAC73325), gspA(AAC76632), ldhA (AAC74462), pflb (AAC73989), adhE (AAC74323), pta(AAC75357), poxB (AAC73958), ackA (AAC75356), and ackB (BAB81430). Theresulting host cells will have increased acetyl-CoA production levelswhen grown in an appropriate environment.

Malonyl-CoA overexpression can be effected by introducing accABCD (e.g.,accession number AAC73296, EC 6.4.1.2) into a host cell. Fatty acids canbe further overexpressed in host cells by introducing into the host cella DNA sequence encoding a lipase (e.g., accession numbers CAA89087,CAA98876).

In addition, inhibiting PlsB can lead to an increase in the levels oflong chain acyl-ACP, which will inhibit early steps in the pathway(e.g., accABCD, fabH, and fabI). The plsB (e.g., accession numberAAC77011) D311E mutation can be used to increase the amount of availableacyl-CoA.

In addition, a host cell can be engineered to overexpress a sfa gene(suppressor of fabA, e.g., accession number AAN79592) to increaseproduction of monounsaturated fatty acids (Rock et al., J. Bacteriology178:5382-5387, 1996).

In some instances, host cells can be engineered to express, overexpress,or attenuate expression of a thioesterase to increase fatty acidsubstrate production. The chain length of a fatty acid substrate iscontrolled by thioesterase. In some instances, a tes or fat gene can beoverexpressed. In other instances, C₁₀ fatty acids can be produced byattenuating thioesterase C₁₈ (e.g., accession numbers AAC73596 andP0ADA1), which uses C_(18:1)-ACP, and expressing thioesterase C₁₀ (e.g.,accession number Q39513), which uses C₁₀-ACP. This results in arelatively homogeneous population of fatty acids that have a carbonchain length of 10. In yet other instances, C₁₄ fatty acids can beproduced by attenuating endogenous thioesterases that produce non-C₁₄fatty acids and expressing the thioesterases, that use C₁₄-ACP (forexample, accession number Q39473). In some situations, C₁₂ fatty acidscan be produced by expressing thioesterases that use C₁₂-ACP (forexample, accession number Q41635) and attenuating thioesterases thatproduce non-C₁₂ fatty acids. Acetyl-CoA, malonyl-CoA, and fatty acidoverproduction can be verified using methods known in the art, forexample, by using radioactive precursors, HPLC, and GC-MS subsequent tocell lysis. Non-limiting examples of thioesterases that can be used inthe methods described herein are listed in Table 2.

TABLE 2 Thioesterases Preferential Accession Number Source Organism Geneproduct produced AAC73596 E. coli tesA without C_(18:1) leader sequenceAAC73555 E. coli tesB Q41635, AAA34215 Umbellularia california fatBC_(12:0) Q39513; AAC49269 Cuphea hookeriana fatB2 C_(8:0)-C_(10:0)AAC49269; AAC72881 Cuphea hookeriana fatB3 C_(14:0)-C_(16:0) Q39473,AAC49151 Cinnamonum camphorum fatB C_(14:0) CAA85388 Arabidopsisthaliana fatB [M141T]* C_(16:1) NP 189147; NP 193041 Arabidopsisthaliana fatA C_(18:1) CAC39106 Bradyrhiizobium japonicum fatA C_(18:1)AAC72883 Cuphea hookeriana fatA C_(18:1) AAL79361 Helianthus annus fatA1*Mayer et al., BMC Plant Biology 7: 1-11, 2007Formation of Branched Aldehydes, Fatty Alcohols, Alkanes, and Alkenes

Aldehydes, fatty alcohols, alkanes, an alkenes can be produced thatcontain branch points by using branched fatty acid derivatives assubstrates. For example, although E. coli naturally produces straightchain fatty acid derivatives (sFAs), E. coli can be engineered toproduce branched chain fatty acid derivatives (brFAs) by introducing andexpressing or overexpressing genes that provide branched precursors inthe E. coli (e.g., bkd, ilv, icm, and fab gene families). Additionally,a host cell can be engineered to express or overexpress genes encodingproteins for the elongation of brFAs (e.g., ACP, FabF, etc.) and/or todelete or attenuate the corresponding host cell genes that normally leadto sFAs.

The first step in forming brFAs is the production of the correspondingα-keto acids by a branched-chain amino acid aminotransferase. Host cellsmay endogenously include genes encoding such enzymes or such genes canbe recombinantly introduced. E. coli, for example, endogenouslyexpresses such an enzyme, IlvE (EC 2.6.1.42; GenBank accessionYP_026247). In some host cells, a heterologous branched-chain amino acidaminotransferase may not be expressed. However, E. coli IlvE or anyother branched-chain amino acid aminotransferase (e.g., IlvE fromLactococcus lactis (GenBank accession AAF34406), IlvE from Pseudomonasputida (GenBank accession NP_745648), or IlvE from Streptomycescoelicolor (GenBank accession NP_629657)), if not endogenous, can beintroduced and recombinantly expressed.

The second step is the oxidative decarboxylation of the α-ketoacids tothe corresponding branched-chain acyl-CoA. This reaction can becatalyzed by a branched-chain α-keto acid dehydrogenase complex (bkd; EC1.2.4.4.) (Denoya et al., J. Bacteriol. 177:3504, 1995), which consistsof E1α/β (decarboxylase), E2 (dihydrolipoyl transacylase), and E3(dihydrolipoyl dehydrogenase) subunits. These branched-chain α-keto aciddehydrogenase complexes are similar to pyruvate and α-ketoglutaratedehydrogenase complexes. Any microorganism that possesses brFAs and/orgrows on branched-chain amino acids can be used as a source to isolatebkd genes for expression in host cells, for example, E. coli.Furthermore, E. coli has the E3 component as part of its pyruvatedehydrogenase complex (lpd, EC 1.8.1.4, GenBank accession NP_414658).Thus, it can be sufficient to express only the E1 α/β and E2 bkd genes.Table 3 lists non-limiting examples of bkd genes from severalmicroorganisms that can be recombinantly introduced and expressed in ahost cell to provide branched-chain acyl-CoA precursors.

TABLE 3 Bkd genes from selected microorganisms GenBank Organism GeneAccession # Streptomyces coelicolor bkdA1 (E1α) NP_628006 bkdB1 (E1β)NP_628005 bkdC1 (E2) NP_638004 Streptomyces coelicolor bkdA2 (E1α)NP_733618 bkdB2 (E1β) NP_628019 bkdC2 (E2) NP_628018 Streptomycesavermitilis bkdA (E1a) BAC72074 bkdB (E1b) BAC72075 bkdC (E2) BAC72076Streptomyces avermitilis bkdF (E1α) BAC72088 bkdG (E1β) BAC72089 bkdH(E2) BAC72090 Bacillus subtilis bkdAA (E1α) NP_390288 bkdAB (E1β)NP_390288 bkdB (E2) NP_390288 Pseudomonas putida bkdA1 (E1α) AAA65614bkdA2 (E1β) AAA65615 bkdC (E2) AAA65617

In another example, isobutyryl-CoA can be made in a host cell, forexample in E. coli, through the coexpression of a crotonyl-CoA reductase(Ccr, EC 1.6.5.5, 1.1.1.1) and isobutyryl-CoA mutase (large subunitIcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (Han and Reynolds,J. Bacteriol. 179:5157, 1997). Crotonyl-CoA is an intermediate in fattyacid biosynthesis in E. coli and other microorganisms. Non-limitingexamples of ccr and icm genes from selected microorganisms are listed inTable 4.

TABLE 4 Ccr and icm genes from selected microorganisms GenBank OrganismGene Accession # Streptomyces coelicolor Ccr NP_630556 icmA NP_629554icmB NP_630904 Streptomyces cinnamonensis ccr AAD53915 icmA AAC08713icmB AJ246005

In addition to expression of the bkd genes, the initiation of brFAbiosynthesis utilizes β-ketoacyl-acyl-carrier-protein synthase III(FabH, EC 2.3.1.41) with specificity for branched chain acyl-CoAs (Li etal., J. Bacteriol. 187:3795-3799, 2005). Non-limiting examples of suchFabH enzymes are listed in Table 5. fabH genes that are involved infatty acid biosynthesis of any brFA-containing microorganism can beexpressed in a host cell. The Bkd and FabH enzymes from host cells thatdo not naturally make brFA may not support brFA production. Therefore,bkd and fabH can be expressed recombinantly. Vectors containing the bkdand fabH genes can be inserted into such a host cell. Similarly, theendogenous level of Bkd and FabH production may not be sufficient toproduce brFA. In this case, they can be overexpressed. Additionally,other components of the fatty acid biosynthesis pathway can be expressedor overexpressed, such as acyl carrier proteins (ACPs) andβ-ketoacyl-acyl-carrier-protein synthase II (fabF, EC 2.3.1.41)(non-limiting examples of candidates are listed in Table 5). In additionto expressing these genes, some genes in the endogenous fatty acidbiosynthesis pathway can be attenuated in the host cell (e.g., the E.coli genes fabH (GenBank accession #NP_415609) and/or fabF (GenBankaccession #NP_415613)).

TABLE 5 FabH, ACP and fabF genes from selected microorganisms with brFAsGenBank Organism Gene Accession # Streptomyces coelicolor fabH1NP_626634 ACP NP_626635 fabF NP_626636 Streptomyces avermitilis fabH3NP_823466 fabC3 (ACP) NP_823467 fabF NP_823468 Bacillus subtilis fabH_ANP_389015 fabH_B NP_388898 ACP NP_389474 fabF NP_389016 StenotrophomonasSmalDRAFT_0818 (FabH) ZP_01643059 maltophilia SmalDRAFT_0821 (ACP)ZP_01643063 SmalDRAFT_0822 (FabF) ZP_01643064 Legionella pneumophilaFabH YP_123672 ACP YP_123675 fabF YP_123676Formation of Cyclic Aldehydes, Fatty Alcohols, Alkanes, and Alkenes

Cyclic aldehydes, fatty alcohols, alkanes, and alkenes can be producedby using cyclic fatty acid derivatives as substrates. To produce cyclicfatty acid derivative substrates, genes that provide cyclic precursors(e.g., the ans, chc, and plm gene families) can be introduced into thehost cell and expressed to allow initiation of fatty acid biosynthesisfrom cyclic precursors. For example, to convert a host cell, such as E.coli, into one capable of synthesizing ω-cyclic fatty acid derivatives(cyFA), a gene that provides the cyclic precursor cyclohexylcarbonyl-CoA(CHC-CoA) (Cropp et al., Nature Biotech. 18:980-983, 2000) can beintroduced and expressed in the host cell. Non-limiting examples ofgenes that provide CHC-CoA in E. coli include: ansJ, ansK, ansL, chcA,and ansM from the ansatrienin gene cluster of Streptomyces collinus(Chen et al., Eur. J. Biochem. 261: 98-107, 1999) or plmJ, plmK, plmL,chcA, and plmM from the phoslactomycin B gene cluster of Streptomycessp. HK803 (Palaniappan et al., J. Biol. Chem. 278:35552-35557, 2003)together with the chcB gene (Patton et al., Biochem. 39:7595-7604, 2000)from S. collinus, S. avermitilis, or S. coelicolor (see Table 6). Thegenes listed in Table 5 can then be expressed to allow initiation andelongation of ω-cyclic fatty acids. Alternatively, the homologous genescan be isolated from microorganisms that make cyFA and expressed in ahost cell (e.g., E. coli).

TABLE 6 Genes for the synthesis of CHC-CoA GenBank Organism GeneAccession # Streptomyces collinus ansJK U72144* ansL chcA ansM chcBAF268489 Streptomyces sp. HK803 pmlJK AAQ84158 pmlL AAQ84159 chcAAAQ84160 pmlM AAQ84161 Streptomyces coelicolor chcB/caiD NP_629292Streptomyces avermitilis chcB/caiD NP_629292 *Only chcA is annotated inGenBank entry U72144, ansJKLM are according to Chen et al. (Eur. J.Biochem. 261: 98-107, 1999).

The genes listed in Table 5 (fabH, ACP, and fabF) allow initiation andelongation of ω-cyclic fatty acid derivatives because they have broadsubstrate specificity. If the coexpression of any of these genes withthe genes listed in Table 6 does not yield cyFA, then fabH, ACP, and/orfabF homologs from microorganisms that make cyFAs (e.g., those listed inTable 7) can be isolated (e.g., by using degenerate PCR primers orheterologous DNA sequence probes) and coexpressed.

TABLE 7 Non-limiting examples of microorganisms that contain ω-cyclicfatty acids Organism Reference Curtobacterium pusillum ATCC19096Alicyclobacillus acidoterrestris ATCC49025 Alicyclobacillusacidocaldarius ATCC27009 Alicyclobacillus cycloheptanicus * Moore, J.Org. Chem. 62: pp. 2173, 1997 * Uses cycloheptylcarbonyl-CoA and notcyclohexylcarbonyl-CoA as precursor for cyFA biosynthesis.Aldehyde, Fatty Alcohol, and Alkene Saturation Levels

The degree of saturation in fatty acid derivatives can be controlled byregulating the degree of saturation of fatty acid derivativeintermediates. The sfa, gns, and fab families of genes can be expressedor overexpressed to control the saturation of fatty acids. FIG. 40 listsnon-limiting examples of genes in these gene families that may be usedin the methods and host cells described herein.

Host cells can be engineered to produce unsaturated fatty acids byengineering the host cell to overexpress fabB or by growing the hostcell at low temperatures (e.g., less than 37° C.). FabB has preferenceto cis-δ3decenoyl-ACP and results in unsaturated fatty acid productionin E. coli. Overexpression of fabB results in the production of asignificant percentage of unsaturated fatty acids (de Mendoza et al., J.Biol. Chem. 258:2098-2101, 1983). The gene fabB may be inserted into andexpressed in host cells not naturally having the gene. These unsaturatedfatty acid derivatives can then be used as intermediates in host cellsthat are engineered to produce fatty acid derivatives, such as fattyaldehydes, fatty alcohols, or alkenes.

In other instances, a repressor of fatty acid biosynthesis, for example,fabR (GenBank accession NP_418398), can be deleted, which will alsoresult in increased unsaturated fatty acid production in E. coli (Zhanget al., J. Biol. Chem. 277:15558, 2002). Similar deletions may be madein other host cells. A further increase in unsaturated fatty acidderivatives may be achieved, for example, by overexpressing fabM(trans-2, cis-3-decenoyl-ACP isomerase, GenBank accession DAA05501) andcontrolled expression of fabK (trans-2-enoyl-ACP reductase II, GenBankaccession NP_357969) from Streptococcus pneumoniae (Marrakchi et al., J.Biol. Chem. 277: 44809, 2002), while deleting E. coli fabI(trans-2-enoyl-ACP reductase, GenBank accession NP_415804). In someexamples, the endogenous fabF gene can be attenuated, thus increasingthe percentage of palmitoleate (C16:1) produced.

Other Substrates

Other substrates that can be used to produce aldehydes, fatty alcohols,alkanes, and alkenes in the methods described herein are acyl-ACP,acyl-CoA, a fatty aldehyde, or a fatty alcohol, which are described in,for example, PCT/US08/058788. Exemplary genes that can be altered toexpress or overexpress these substrates in host cells are listed in FIG.40. Other exemplary genes are described in PCT/US08/058788.

Genetic Engineering of Host Cells to Produce Aldehydes, Fatty Alcohols,Alkanes, and Alkenes

Various host cells can be used to produce aldehydes, fatty alcohols,alkanes, and/or alkenes, as described herein. A host cell can be anyprokaryotic or eukaryotic cell. For example, a polypeptide describedherein can be expressed in bacterial cells (such as E. coli), insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) cells, COS cells, VERO cells, BHK cells, HeLa cells, Cv1 cells,MDCK cells, 293 cells, 3T3 cells, or PC12 cells). Other exemplary hostcells include cells from the members of the genus Escherichia, Bacillus,Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma,Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia,Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes,Chrysosporium, Saccharomyces, Schizosaccharomyces, Yarrowia, orStreptomyces. Yet other exemplary host cells can be a Bacillus lentuscell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, aBacillus licheniformis cell, a Bacillus alkalophilus cell, a Bacilluscoagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, aBacillus thuringiensis cell, a Bacillus clausii cell, a Bacillusmegaterium cell, a Bacillus subtilis cell, a Bacillus amyloliquefacienscell, a Trichoderma koningii cell, a Trichoderma viride cell, aTrichoderma reesei cell, a Trichoderma longibrachiatum cell, anAspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillusfoetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell,an Aspergillus oryzae cell, a Humicola insolens cell, a Humicolalanuginose cell, a Rhizomucor miehei cell, a Mucor michei cell, aStreptomyces lividans cell, a Streptomyces murinus cell, or anActinomycetes cell.

Other nonlimiting examples of host cells are those listed in Table 1.

In a preferred embodiment, the host cell is an E. coli cell. In a morepreferred embodiment, the host cell is from E. coli strains B, C, K, orW. Other suitable host cells are known to those skilled in the art.

Various methods well known in the art can be used to geneticallyengineer host cells to produce aldehydes, fatty alcohols, alkanes and/oralkenes. The methods include the use of vectors, preferably expressionvectors, containing a nucleic acid encoding an aldehyde, fatty alcohol,alkane, and/or alkene biosynthetic polypeptide described herein, or apolypeptide variant or fragment thereof. As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell and arethereby replicated along with the host genome. Moreover, certainvectors, such as expression vectors, are capable of directing theexpression of genes to which they are operatively linked. In general,expression vectors used in recombinant DNA techniques are often in theform of plasmids. However, other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenoviruses,and adeno-associated viruses), can also be used.

The recombinant expression vectors described herein include a nucleicacid described herein in a form suitable for expression of the nucleicacid in a host cell. The recombinant expression vectors can include oneor more control sequences, selected on the basis of the host cell to beused for expression. The control sequence is operably linked to thenucleic acid sequence to be expressed. Such control sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Controlsequences include those that direct constitutive expression of anucleotide sequence in many types of host cells and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectorsdescribed herein can be introduced into host cells to producepolypeptides, including fusion polypeptides, encoded by the nucleicacids as described herein.

Recombinant expression vectors can be designed for expression of analdehyde, fatty alcohol, alkane, and/or alkene biosynthetic polypeptideor variant in prokaryotic or eukaryotic cells (e.g., bacterial cells,such as E. coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells). Suitable host cells are discussedfurther in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example, by using T7 promoter regulatory sequences and T7polymerase.

Expression of polypeptides in prokaryotes, for example, E. coli, is mostoften carried out with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionpolypeptides. Fusion vectors add a number of amino acids to apolypeptide encoded therein, usually to the amino terminus of therecombinant polypeptide. Such fusion vectors typically serve threepurposes: (1) to increase expression of the recombinant polypeptide; (2)to increase the solubility of the recombinant polypeptide; and (3) toaid in the purification of the recombinant polypeptide by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant polypeptide. This enables separation of therecombinant polypeptide from the fusion moiety after purification of thefusion polypeptide. Examples of such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin, and enterokinase.Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith et al., Gene (1988) 67:31-40), pMAL (New England Biolabs, Beverly,Mass.), and pRITS (Pharmacia, Piscataway, N.J.), which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant polypeptide.

Examples of inducible, non-fusion E. coli expression vectors includepTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d (Studier et al.,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990) 60-89). Target gene expression from the pTrcvector relies on host RNA polymerase transcription from a hybrid trp-lacfusion promoter. Target gene expression from the pET 11d vector relieson transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression is toexpress the polypeptide in a host cell with an impaired capacity toproteolytically cleave the recombinant polypeptide (see Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the host cell (Wada et al., Nucleic Acids Res. (1992) 20:2111-2118).Such alteration of nucleic acid sequences can be carried out by standardDNA synthesis techniques.

In another embodiment, the host cell is a yeast cell. In thisembodiment, the expression vector is a yeast expression vector. Examplesof vectors for expression in yeast S. cerevisiae include pYepSec1(Baldari et al., EMBO J. (1987) 6:229-234), pMFa (Kurjan et al., Cell(1982) 30:933-943), pJRY88 (Schultz et al., Gene (1987) 54:113-123),pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InvitrogenCorp, San Diego, Calif.).

Alternatively, a polypeptide described herein can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include, for example, the pAc series (Smith et al., Mol. CellBiol. (1983) 3:2156-2165) and the pVL series (Lucklow et al., Virology(1989) 170:31-39).

In yet another embodiment, the nucleic acids described herein can beexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, Nature(1987) 329:840) and pMT2PC (Kaufman et al., EMBO J. (1987) 6:187-195).When used in mammalian cells, the expression vector's control functionscan be provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. Other suitable expression systems for both prokaryoticand eukaryotic cells are described in chapters 16 and 17 of Sambrook etal., eds., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

Vectors can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in, for example, Sambrook et al. (supra).

For stable transformation of bacterial cells, it is known that,depending upon the expression vector and transformation technique used,only a small fraction of cells will take-up and replicate the expressionvector. In order to identify and select these transformants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) can beintroduced into the host cells along with the gene of interest.Selectable markers include those that confer resistance to drugs, suchas ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a polypeptide described herein or canbe introduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) can be introducedinto the host cells along with the gene of interest. Preferredselectable markers include those which confer resistance to drugs, suchas G418, hygromycin, and methotrexate. Nucleic acids encoding aselectable marker can be introduced into a host cell on the same vectoras that encoding a polypeptide described herein or can be introduced ona separate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

In certain methods, an aldehyde biosynthetic polypeptide and an alkaneor alkene biosynthetic polypeptide are co-expressed in a single hostcell. In alternate methods, an aldehyde biosynthetic polypeptide and analcohol dehydrogenase polypeptide are co-expressed in a single hostcell.

Transport Proteins

Transport proteins can export polypeptides and hydrocarbons (e.g.,aldehydes, alkanes, and/or alkenes) out of a host cell. Many transportand efflux proteins serve to excrete a wide variety of compounds and canbe naturally modified to be selective for particular types ofhydrocarbons.

Non-limiting examples of suitable transport proteins are ATP-BindingCassette (ABC) transport proteins, efflux proteins, and fatty acidtransporter proteins (FATP). Additional non-limiting examples ofsuitable transport proteins include the ABC transport proteins fromorganisms such as Caenorhabditis elegans, Arabidopsis thalania,Alkaligenes eutrophus, and Rhodococcus erythropolis. Exemplary ABCtransport proteins that can be used are listed in FIG. 40 (e.g., CER5,AtMRP5, AmiS2, and AtPGP1). Host cells can also be chosen for theirendogenous ability to secrete hydrocarbons. The efficiency ofhydrocarbon production and secretion into the host cell environment(e.g., culture medium, fermentation broth) can be expressed as a ratioof intracellular product to extracellular product. In some examples, theratio can be about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

Fermentation

The production and isolation of aldehydes, fatty alcohols, alkanesand/or alkenes can be enhanced by employing beneficial fermentationtechniques. One method for maximizing production while reducing costs isincreasing the percentage of the carbon source that is converted tohydrocarbon products.

During normal cellular lifecycles, carbon is used in cellular functions,such as producing lipids, saccharides, proteins, organic acids, andnucleic acids. Reducing the amount of carbon necessary forgrowth-related activities can increase the efficiency of carbon sourceconversion to product. This can be achieved by, for example, firstgrowing host cells to a desired density (for example, a density achievedat the peak of the log phase of growth). At such a point, replicationcheckpoint genes can be harnessed to stop the growth of cells.Specifically, quorum sensing mechanisms (reviewed in Camilli et al.,Science 311:1113, 2006; Venturi FEMS Microbio. Rev. 30:274-291, 2006;and Reading et al., FEMS Microbiol. Lett. 254:1-11, 2006) can be used toactivate checkpoint genes, such as p53, p21, or other checkpoint genes.

Genes that can be activated to stop cell replication and growth in E.coli include umuDC genes. The overexpression of umuDC genes stops theprogression from stationary phase to exponential growth (Murli et al.,J. of Bact. 182:1127, 2000). UmuC is a DNA polymerase that can carry outtranslesion synthesis over non-coding lesions—the mechanistic basis ofmost UV and chemical mutagenesis. The umuDC gene products are involvedin the process of translesion synthesis and also serve as a DNA sequencedamage checkpoint. The umuDC gene products include UmuC, UmuD, umuD′,UmuD′₂C, UmuD′₂, and UmuD₂. Simultaneously, product-producing genes canbe activated, thus minimizing the need for replication and maintenancepathways to be used while an aldehyde, alkane and/or alkene is beingmade. Host cells can also be engineered to express umuC and umuD from E.coli in pBAD24 under the prpBCDE promoter system through de novosynthesis of this gene with the appropriate end-product productiongenes.

The percentage of input carbons converted to aldehydes, fatty alcohols,alkanes and/or alkenes can be a cost driver. The more efficient theprocess is (i.e., the higher the percentage of input carbons convertedto aldehydes, fatty alcohols, alkanes and/or alkenes), the lessexpensive the process will be. For oxygen-containing carbon sources(e.g., glucose and other carbohydrate based sources), the oxygen must bereleased in the form of carbon dioxide. For every 2 oxygen atomsreleased, a carbon atom is also released leading to a maximaltheoretical metabolic efficiency of approximately 34% (w/w) (for fattyacid derived products). This figure, however, changes for otherhydrocarbon products and carbon sources. Typical efficiencies in theliterature are approximately less than 5%. Host cells engineered toproduce aldehydes, alkanes and/or alkenes can have greater than about 1,3, 5, 10, 15, 20, 25, and 30% efficiency. In one example, host cells canexhibit an efficiency of about 10% to about 25%. In other examples, suchhost cells can exhibit an efficiency of about 25% to about 30%. In otherexamples, host cells can exhibit greater than 30% efficiency.

The host cell can be additionally engineered to express recombinantcellulosomes, such as those described in PCT application numberPCT/US2007/003736. These cellulosomes can allow the host cell to usecellulosic material as a carbon source. For example, the host cell canbe additionally engineered to express invertases (EC 3.2.1.26) so thatsucrose can be used as a carbon source. Similarly, the host cell can beengineered using the teachings described in U.S. Pat. Nos. 5,000,000;5,028,539; 5,424,202; 5,482,846; and 5,602,030; so that the host cellcan assimilate carbon efficiently and use cellulosic materials as carbonsources.

In one example, the fermentation chamber can enclose a fermentation thatis undergoing a continuous reduction. In this instance, a stablereductive environment can be created. The electron balance can bemaintained by the release of carbon dioxide (in gaseous form). Effortsto augment the NAD/H and NADP/H balance can also facilitate instabilizing the electron balance. The availability of intracellularNADPH can also be enhanced by engineering the host cell to express anNADH:NADPH transhydrogenase. The expression of one or more NADH:NADPHtranshydrogenases converts the NADH produced in glycolysis to NADPH,which can enhance the production of aldehydes, alkanes and/or alkenes.

For small scale production, the engineered host cells can be grown inbatches of, for example, around 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L;fermented; and induced to express desired aldehydes, fatty alcohols,alkanes and/or alkenes based on the specific genes encoded in theappropriate plasmids. For example, E. coli BL21(DE3) cells harboringpBAD24 (with ampicillin resistance and the aldehyde, fatty alcohol,alkane, or alkene synthesis pathway) as well as pUMVC1 (with kanamycinresistance and the acetyl CoA/malonyl CoA overexpression system) can beincubated overnight in 2 L flasks at 37° C. shaken at >200 rpm in 500 mLLB medium supplemented with 75 μg/mL ampicillin and 50 μg/mL kanamycinuntil cultures reach an OD₆₀₀ of >0.8. Upon achieving an OD₆₀₀ of >0.8,the cells can be supplemented with 25 mM sodium proprionate (pH 8.0) toactivate the engineered gene systems for production and to stop cellularproliferation by activating UmuC and UmuD proteins. Induction can beperformed for 6 hrs at 30° C. After incubation, the media can beexamined for aldehydes, fatty alcohols, alkanes and/or alkenes usingGC-MS.

For large scale production, the engineered host cells can be grown inbatches of 10 L, 100 L, 1000 L, or larger; fermented; and induced toexpress desired aldehydes, fatty alcohols, alkanes and/or alkenes basedon the specific genes encoded in the appropriate plasmids. For example,E. coli BL21(DE3) cells harboring pBAD24 (with ampicillin resistance andthe aldehyde and/or alkane synthesis pathway) as well as pUMVC1 (withkanamycin resistance and the acetyl-CoA/malonyl-CoA overexpressionsystem) can be incubated from a 500 mL seed culture for 10 Lfermentations (5 L for 100 L fermentations, etc.) in LB media (glycerolfree) with 50 μg/mL kanamycin and 75 μg/mL ampicillin at 37° C., andshaken at >200 rpm until cultures reach an OD₆₀₀ of >0.8 (typically 16hrs). Media can be continuously supplemented to maintain 25 mM sodiumproprionate (pH 8.0) to activate the engineered gene systems forproduction and to stop cellular proliferation by activating umuC andumuD proteins. Media can be continuously supplemented with glucose tomaintain a concentration 25 g/100 mL.

After the first hour of induction, aliquots of no more than 10% of thetotal cell volume can be removed each hour and allowed to sit withoutagitation to allow the aldehydes, alkanes and/or alkenes to rise to thesurface and undergo a spontaneous phase separation. The aldehyde, fattyalcohols, alkane and/or alkene component can then be collected, and theaqueous phase returned to the reaction chamber. The reaction chamber canbe operated continuously. When the OD₆₀₀ drops below 0.6, the cells canbe replaced with a new batch grown from a seed culture.

Producing Aldehydes, Fatty Alcohols, Alkanes and Alkenes Using Cell-FreeMethods

In some methods described herein, an aldehyde, fatty alcohols, alkaneand/or alkene can be produced using a purified polypeptide describedherein and a substrate described herein. For example, a host cell can beengineered to express aldehyde, fatty alcohols, alkane and/or alkenebiosynthetic polypeptide or variant as described herein. The host cellcan be cultured under conditions suitable to allow expression of thepolypeptide. Cell free extracts can then be generated using knownmethods. For example, the host cells can be lysed using detergents or bysonication. The expressed polypeptides can be purified using knownmethods. After obtaining the cell free extracts, substrates describedherein can be added to the cell free extracts and maintained underconditions to allow conversion of the substrates to aldehydes, fattyalcohols, alkanes and/or alkenes. The aldehydes, fatty alcohols, alkanesand/or alkenes can then be separated and purified using knowntechniques.

Post-Production Processing

The aldehydes, fatty alcohols, alkanes and/or alkenes produced duringfermentation can be separated from the fermentation media. Any knowntechnique for separating aldehydes, fatty alcohols, alkanes and/oralkenes from aqueous media can be used. One exemplary separation processis a two phase (bi-phasic) separation process. This process involvesfermenting the genetically engineered host cells under conditionssufficient to produce an aldehyde, fatty alcohols, alkane and/or alkene,allowing the aldehyde, fatty alcohols, alkane and/or alkene to collectin an organic phase, and separating the organic phase from the aqueousfermentation broth. This method can be practiced in both a batch andcontinuous fermentation setting.

Bi-phasic separation uses the relative immiscibility of aldehydes, fattyalcohols, alkanes and/or alkenes to facilitate separation Immisciblerefers to the relative inability of a compound to dissolve in water andis defined by the compound's partition coefficient. One of ordinaryskill in the art will appreciate that by choosing a fermentation brothand organic phase, such that the aldehyde, alkane and/or alkene beingproduced has a high log P value, the aldehyde, alkane and/or alkene canseparate into the organic phase, even at very low concentrations, in thefermentation vessel.

The aldehydes, fatty alcohols, alkanes and/or alkenes produced by themethods described herein can be relatively immiscible in thefermentation broth, as well as in the cytoplasm. Therefore, thealdehyde, fatty alcohols, alkane and/or alkene can collect in an organicphase either intracellularly or extracellularly. The collection of theproducts in the organic phase can lessen the impact of the aldehyde,fatty alcohols, alkane and/or alkene on cellular function and can allowthe host cell to produce more product.

The methods described herein can result in the production of homogeneouscompounds wherein at least about 60%, 70%, 80%, 90%, or 95% of thealdehydes, fatty alcohols, alkanes and/or alkenes produced will havecarbon chain lengths that vary by less than about 6 carbons, less thanabout 4 carbons, or less than about 2 carbons. These compounds can alsobe produced with a relatively uniform degree of saturation. Thesecompounds can be used directly as fuels, fuel additives, specialtychemicals, starting materials for production of other chemical compounds(e.g., polymers, surfactants, plastics, textiles, solvents, adhesives,etc.), or personal care product additives. These compounds can also beused as feedstock for subsequent reactions, for example, hydrogenation,catalytic cracking (via hydrogenation, pyrolisis, or both), to makeother products.

In some embodiments, the aldehydes, fatty alcohols, alkanes and/oralkenes produced using methods described herein can contain betweenabout 50% and about 90% carbon; or between about 5% and about 25%hydrogen. In other embodiments, the aldehydes, fatty alcohols, alkanesand/or alkenes produced using methods described herein can containbetween about 65% and about 85% carbon; or between about 10% and about15% hydrogen.

Fuel Compositions and Specialty Chemical Compositions

The aldehydes, fatty alcohols, alkanes and/or alkenes described hereincan be used as or converted into a fuel or as a specialty chemical. Oneof ordinary skill in the art will appreciate that, depending upon theintended purpose of the fuel or specialty chemical, different aldehydes,fatty alcohols, alkanes and/or alkenes can be produced and used. Forexample, a branched aldehyde, fatty alcohol, alkane and/or alkene may bedesirable for automobile fuel that is intended to be used in coldclimates. In addition, when the aldehydes, fatty alcohols, alkanesand/or alkenes described herein are used as a feedstock for fuel orspecialty chemical production, one of ordinary skill in the art willappreciate that the characteristics of the aldehyde, fatty alcohol,alkane and/or alkene feedstock will affect the characteristics of thefuel or specialty chemical produced. Hence, the characteristics of thefuel or specialty chemical product can be selected for by producingparticular aldehydes, fatty alcohols, alkanes and/or alkenes for use asa feedstock.

Using the methods described herein, biofuels having desired fuelqualities can be produced from aldehydes, fatty alcohols, alkanes and/oralkenes. Biologically produced aldehydes, fatty alcohols, alkanes and/oralkenes represent a new source of biofuels, which can be used as jetfuel, diesel, or gasoline. Some biofuels made using aldehydes, fattyalcohols, alkanes and/or alkenes have not been produced from renewablesources and are new compositions of matter. These new fuels or specialtychemicals can be distinguished from fuels or specialty chemicals derivedfrom petrochemical carbon on the basis of dual carbon-isotopicfingerprinting. Additionally, the specific source of biosourced carbon(e.g., glucose vs. glycerol) can be determined by dual carbon-isotopicfingerprinting (see, e.g., U.S. Pat. No. 7,169,588, in particular col.4, line 31, to col. 6, line 8).

The aldehydes, fatty alcohols, alkanes and/or alkenes and the associatedbiofuels, specialty chemicals, and mixtures can be distinguished fromtheir petrochemical derived counterparts on the basis of ¹⁴C (f_(M)) anddual carbon-isotopic fingerprinting. In some examples, the aldehyde,fatty alcohol, alkane and/or alkene in the biofuel composition can havea fraction of modern carbon (f_(M) ¹⁴C) of, for example, at least about1.003, 1.010, or 1.5.

In some examples, a biofuel composition can be made that includesaldehydes, fatty alcohols, alkanes and/or alkenes having δ¹³C of fromabout −15.4 to about −10.9, where the aldehydes, fatty alcohols, alkanesand/or alkenes account for at least about 85% of biosourced material(i.e., derived from a renewable resource, such as biomass, cellulosicmaterials, and sugars) in the composition.

The ability to distinguish these biologically derived products isbeneficial in tracking these materials in commerce. For example, fuelsor specialty chemicals comprising both biologically derived andpetroleum-based carbon isotope profiles can be distinguished from fuelsand specialty chemicals made only of petroleum-based materials. Thus,the aldehydes, fatty alcohols, alkanes and/or alkenes described hereincan be followed in commerce or identified in commerce as a biofuel onthe basis of their unique profile. In addition, other competingmaterials can be identified as being biologically derived or derivedfrom a petrochemical source.

Fuel additives are used to enhance the performance of a fuel or engine.For example, fuel additives can be used to alter the freezing/gellingpoint, cloud point, lubricity, viscosity, oxidative stability, ignitionquality, octane level, and/or flash point. In the United States, allfuel additives must be registered with Environmental Protection Agency.The names of fuel additives and the companies that sell the fueladditives are publicly available by contacting the EPA or by viewing theagency's website. One of ordinary skill in the art will appreciate thatthe aldehyde- and/or alkane-based biofuels described herein can be mixedwith one or more fuel additives to impart a desired quality.

The aldehyde, fatty alcohols, alkane and/or alkene-based biofuelsdescribed herein can be mixed with other fuels, such as variousalcohols, such as ethanol and butanol, and petroleum-derived products,such as gasoline, diesel, or jet fuel.

In some examples, the mixture can include at least about 10%, 15%, 20%,30%, 40%, 50%, or 60% by weight of the aldehyde, fatty alcohols, alkane,or alkene. In other examples, a biofuel composition can be made thatincludes at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,90% or 95% of an aldehyde, fatty alcohols, alkane, or alkene thatincludes a carbon chain that is 8, 10, 12, 13, 14, 15, 16, 17, 18, 19,20, 21 or 22 carbons in length. Such biofuel compositions canadditionally include at least one additive selected from a cloud pointlowering additive that can lower the cloud point to less than about 5°C., or 0° C.; a surfactant; a microemulsion; at least about 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% diesel fuelfrom triglycerides; petroleum-derived gasoline; or diesel fuel frompetroleum.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Detection and Verification of Alkane Biosynthesis in SelectedCyanobacteria

Seven cyanobacteria, whose complete genome sequences are publiclyavailable, were selected for verification and/or detection of alkanebiosynthesis: Synechococcus elongatus PCC7942, Synechococcus elongatusPCC6301, Anabaena variabilis ATCC29413, Synechocystis sp. PCC6803,Nostoc punctiforme PCC73102, Gloeobacter violaceus ATCC 29082, andProchlorococcus marinus CCMP1986. Only the first three cyanobacterialstrains from this list had previously been reported to contain alkanes(Han et al., J. Am. Chem. Soc. 91:5156-5159 (1969); Fehler et al.,Biochem. 9:418-422 (1970)). The strains were grown photoautotrophicallyin shake flasks in 100 mL of the appropriate media (listed in Table 8)for 3-7 days at 30° C. at a light intensity of approximately 3,500 lux.Cells were extracted for alkane detection as follows: cells from 1 mLculture volume were centrifuged for 1 min at 13,000 rpm, the cellpellets were resuspended in methanol, vortexed for 1 min and thensonicated for 30 min. After centrifugation for 3 min at 13,000 rpm, thesupernatants were transferred to fresh vials and analyzed by GC-MS. Thesamples were analyzed on either 30 m DP-5 capillary column (0.25 mminternal diameter) or a 30 m high temperature DP-5 capillary column(0.25 mm internal diameter) using the following method.

After a 1 μL splitless injection (inlet temperature held at 300° C.)onto the GC/MS column, the oven was held at 100° C. for 3 mins. Thetemperature was ramped up to 320° C. at a rate of 20° C./min. The ovenwas held at 320° C. for an additional 5 min. The flow rate of thecarrier gas helium was 1.3 mL/min. The MS quadrapole scanned from 50 to550 m/z. Retention times and fragmentation patterns of product peakswere compared with authentic references to confirm peak identity.

Out of the seven strains, six produced mainly heptadecane and oneproduced pentadecane (P. marinus CCMP1986); one of these strainsproduced methyl-heptadecane in addition to heptadecane (A. variabilisATCC29413) (see Table 8). Therefore, alkane biosynthesis in threepreviously reported cyanobacteria was verified, and alkane biosynthesiswas detected in four cyanobacteria that were not previously known toproduce alkanes: P. marinus CCMP1986 (see FIG. 1), N. punctiformePCC73102 (see FIG. 2), G. violaceus ATCC 29082 (see FIG. 3) andSynechocystis sp. PCC6803 (see FIG. 4).

FIG. 1A depicts the GC/MS trace of Prochlorococcus marinus CCMP1986cells extracted with methanol. The peak at 7.55 min had the sameretention time as pentadecane (Sigma). In FIG. 1B, the massfragmentation pattern of the pentadecane peak is shown. The 212 peakcorresponds to the molecular weight of pentadecane.

FIG. 2A depicts the GC/MS trace of Nostoc punctiforme PCC73102 cellsextracted with methanol. The peak at 8.73 min has the same retentiontime as heptadecane (Sigma). In FIG. 2B, the mass fragmentation patternof the heptadecane peak is shown. The 240 peak corresponds to themolecular weight of heptadecane.

FIG. 3A depicts the GC/MS trace of Gloeobaceter violaceus ATCC29082cells extracted with methanol. The peak at 8.72 min has the sameretention time as heptadecane (Sigma). In FIG. 3B, the massfragmentation pattern of the heptadecane peak is shown. The 240 peakcorresponds to the molecular weight of heptadecane.

FIG. 4A depicts the GC/MS trace of Synechocystic sp. PCC6803 cellsextracted with methanol. The peak at 7.36 min has the same retentiontime as heptadecane (Sigma). In FIG. 4B, the mass fragmentation patternof the heptadecane peak is shown. The 240 peak corresponds to themolecular weight of heptadecane.

TABLE 8 Hydrocarbons detected in selected cyanobacteria AlkanesCyanobacterium ATCC# Genome Medium reported verified ² Synechococcuselongatus PCC7942 27144 2.7 Mb BG-11 C17:0 C17:0, C15:0 Synechococcuselongatus PCC6301 33912 2.7 Mb BG-11 C17:0 C17:0, C15:0 Anabaenavariabilis 29413 6.4 Mb BG-11 C17:0, 7- or 8-Me-C17:0 C17:0, Me-C17:0Synechocystis sp. PCC6803 27184 3.5 Mb BG-11 — C17:0, C15:0Prochlorococcus marinus CCMP1986 ¹ — 1.7 Mb — — C15:0 Nostoc punctiformePCC73102 29133 9.0 Mb ATCC819 — C17:0 Gloeobacter violaceus 29082 4.6 MbBG11 — C17:0 ¹ cells for extraction were a gift from Jacob Waldbauer(MIT) ² major hydrocarbon is in bold

Genomic analysis yielded two genes that were present in thealkane-producing strains. The Synechococcus elongatus PCC7942 homologsof these genes are depicted in Table 9 and are Synpcc7942_1593 (SEQ IDNO:1) and Synpcc7942_1594 (SEQ ID NO:65).

TABLE 9 Alkane-producing cyanobacterial genes Gene Object Genbank IDLocus Tag accession Gene Name Length COG Pfam InterPro Notes 637800026Synpcc7942_1593 YP_400610 hypothetical protein 231 aa — pfam02915IPR009078 ferritin/ribonucleotide IPR003251 reductase-like rubreryhtrin637800027 Synpcc7942_1594 YP_400611 hypothetical protein 341 aa COG5322pfam00106 IPR000408 predicted dehydrogenase IPR016040 NAD(P)-bindingIPR002198 short chain dehydrogenase

Example 2 Deletion of the sll0208 and sll0209 genes in Synechocystis sp.PCC6803 Leads to Loss of Alkane Biosynthesis

The genes encoding the putative decarbonylase (sll0208; NP_442147) (SEQID NO:3) and aldehyde-generating enzyme (sll0209; NP_442146) (SEQ IDNO:67) of Synechocystis sp. PCC6803 were deleted as follows.Approximately 1 kb of upstream and downstream flanking DNA wereamplified using primer sll0208/9-KO1 (CGCGGATCCCTTGATTCTACTGCGGCGAGT)with primer sll0208/9-KO2(CACGCACCTAGGTTCACACTCCCATGGTATAACAGGGGCGTTGGACTCC TGTG) and primersll0208/9-KO3 (GTTATACCATGGGAGTGTGAACCTAGGTGCGTGGCCGACAGGATAGGGCGTGT)with primer sll0208/9-KO4 (CGCGGATCCAACGCATCCTCACTAGTCGGG),respectively. The PCR products were used in a cross-over PCR withprimers sll0208/9-KO1 and sll0208/9-KO4 to amplify the approximately 2kb sll0208/sll0209 deletion cassette, which was cloned into the BamHIsite of the cloning vector pUC19. A kanamycin resistance cassette (aph,KanR) was then amplified from plasmid pRL27 (Larsen et al., Arch.Microbiol. 178:193 (2002)) using primers Kan-aph-F(CATGCCATGGAAAGCCACGTTGTGTCTCAAAATCTCTG) and Kan-aph-R(CTAGTCTAGAGCGCTGAGGTCTGCCTCGTGAA), which was then cut with NcoI andXbaI and cloned into the NcoI and AvrII sites of the sll0208/sll0209deletion cassette, creating a sll0208/sll0209-deletion KanR-insertioncassette in pUC19. The cassette-containing vector, which does notreplicate in cyanobacteria, was transformed into Synechocystis sp.PCC6803 (Zang et al., 2007, J. Microbiol., vol. 45, pp. 241) andtransformants (e.g., chromosomal integrants by double-homologousrecombination) were selected on BG-11 agar plates containing 100 μg/mLKanamycin in a light-equipped incubator at 30° C. Kanamycin resistantcolonies were restreaked once and then subjected to genotypic analysisusing PCR with diagnostic primers.

Confirmed deletion-insertion mutants were cultivated in 12 mL of BG11medium with 50 μg/mL Kanamycin for 4 days at 30° C. in a light-equippedshaker-incubator. 1 mL of broth was then centrifuged (1 min at 13,000 g)and the cell pellets were extracted with 0.1 mL methanol. Afterextraction, the samples were again centrifuged and the supernatants weresubjected to GC-MS analysis as described in Example 1.

As shown in FIG. 5, the Synechocystis sp. PCC6803 strains in which thesll0208 and sll0209 genes were deleted lost their ability to produceheptadecene and octadecenal. This result demonstrates that the sll0208and sll0209 genes in Synechocystis sp. PCC6803 and the orthologous genesin other cyanobacteria (see Table 1) are responsible for alkane andfatty aldehyde biosynthesis in these organisms.

Example 3 Production of Fatty Aldehydes and Fatty Alcohols in E. coliThrough Heterologous Expression of Synechococcus elongatus PCC7942orf1594

The genomic DNA encoding Synechococcus elongatus PCC7942 orf1594(YP_400611; putative aldehyde-generating enzyme) (SEQ ID NO:65) wasamplified and cloned into the NcoI and EcoRI sites of vector OP-80(pCL1920 derivative) under the control of the P_(trc) promoter. Theresulting construct (“OP80-PCC7942_1594”) was transformed into E. coliMG1655 and the cells were grown at 37° C. in M9 minimal media with 1%(w/v) glucose as carbon source and supplemented with 100 μg/mLspectinomycin. When the culture reached OD₆₀₀ of 0.8-1.0, it was inducedwith 1 mM IPTG and cells were grown for an additional 18-20 h at 37° C.Cells from 0.5 mL of culture were extracted with 0.5 mL of ethylacetate. After sonication for 60 min, the sample was centrifuged at15,000 rpm for 5 min. The solvent layer was analyzed by GC-MS asdescribed in Example 1.

As shown in FIG. 6, E. coli cells transformed with the Synechococcuselongatus PCC7942 orf1594-bearing vector produced the following fattyaldehydes and fatty alcohols: hexadecanal, octadecenal, tetradecenol,hexadecenol, hexadecanol and octadecenol. This result indicates thatPCC7942 orf1594 (i) generates aldehydes in-vivo as possible substratesfor decarbonylation and (ii) may reduce acyl-ACPs as substrates, whichare the most abundant form of activated fatty acids in wild type E. colicells. Therefore, the enzyme was named Acyl-ACP reductase. In-vivo, thefatty aldehydes apparently are further reduced to the correspondingfatty alcohols by an endogenous E. coli aldehyde reductase activity.

Example 4 Production of Fatty Aldehydes and Fatty Alcohols in E. coliThrough Heterologous Expression of Cyanothece sp. ATCC51142 cce_1430

The genomic DNA encoding Cyanothece sp. ATCC51142 cce_1430(YP_001802846; putative aldehyde-generating enzyme) (SEQ ID NO:69) wasamplified and cloned into the NcoI and EcoRI sites of vector OP-80(pCL1920 derivative) under the control of the P_(trc) promoter. Theresulting construct was transformed into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media with 1% (w/v) glucose as carbonsource and supplemented with 100 μg/mL spectinomycin. The cells werecultured and extracted as in Example 3 and analyzed by GC-MS asdescribed in Example 26.

As shown in FIG. 7, E. coli cells transformed with the Cyanothece sp.ATCC51142 cce_1430-bearing vector produced the following fatty aldehydesand fatty alcohols: hexadecanal, octadecenal, tetradecenol, hexadecenol,hexadecanol and octadecenol. This result indicates that ATCC51142cce_1430 (i) generates aldehydes in-vivo as possible substrates fordecarbonylation and (ii) may reduce acyl-ACPs as substrates, which arethe most abundant form of activated fatty acids in wild type E. colicells. Therefore, this enzyme is also an Acyl-ACP reductase.

Example 5 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andSynechococcus elongatus PCC7942 orf1593

The genomic DNA encoding Synechococcus elongatus PCC7942 orf1593(YP_400610; putative decarbonylase) (SEQ ID NO:1) was amplified andcloned into the NdeI and XhoI sites of vector OP-183 (pACYC derivative)under the control of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example1.

As shown in FIG. 8, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and S. elongatus PCC7942_1593-bearing vectors produced thesame fatty aldehydes and fatty alcohols as in Example 3, but alsopentadecane and heptadecene. This result indicates that PCC7942_1593 inE. coli converts hexadecanal and octadecenal to pentadecane andheptadecene, respectively, and therefore is an active fatty aldehydedecarbonylase.

Example 6 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andNostoc punctiforme PCC73102 Npun02004178

The genomic DNA encoding Nostoc punctiforme PCC73102 Npun02004178(ZP_00108838; putative decarbonylase) (SEQ ID NO:5) was amplified andcloned into the NdeI and XhoI sites of vector OP-183 (pACYC derivative)under the control of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example1.

As shown in FIG. 9, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and N. punctiforme PCC73102 Npun02004178-bearing vectorsproduced the same fatty aldehydes and fatty alcohols as in Example 3,but also tridecane, pentadecene, pentadecane and heptadecene. Thisresult indicates that Npun02004178 in E. coli converts tetradecanal,hexadecenal, hexadecanal and octadecenal to tridecane, pentadecene,pentadecane and heptadecene, respectively, and therefore is an activefatty aldehyde decarbonylase.

Example 7 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andSynechocystis sp. PCC6803 sll0208

The genomic DNA encoding Synechocystis sp. PCC6803 sll0208 (NP_442147;putative decarbonylase) (SEQ ID NO:3) was amplified and cloned into theNdeI and XhoI sites of vector OP-183 (pACYC derivative) under thecontrol of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example1.

As shown in FIG. 10, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and Synechocystis sp. PCC6803 sll0208-bearing vectorsproduced the same fatty aldehydes and fatty alcohols as in Example 3,but also pentadecane and heptadecene. This result indicates thatNpun02004178 in E. coli converts hexadecanal and octadecenal topentadecane and heptadecene, respectively, and therefore is an activefatty aldehyde decarbonylase.

Example 8 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andNostoc sp. PCC7210 alr5283

The genomic DNA encoding Nostoc sp. PCC7210 alr5283 (NP_489323; putativedecarbonylase) (SEQ ID NO:7) was amplified and cloned into the NdeI andXhoI sites of vector OP-183 (pACYC derivative) under the control of theP_(trc) promoter. The resulting construct was cotransformed withOP80-PCC7942_1594 into E. coli MG1655 and the cells were grown at 37° C.in M9 minimal media supplemented with 100 μg/mL spectinomycin and 100μg/mL carbenicillin. The cells were cultured and extracted as in Example3 and analyzed by GC-MS as described in Example 1.

As shown in FIG. 11, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and Nostoc sp. PCC7210 alr5283-bearing vectors produced thesame fatty aldehydes and fatty alcohols as in Example 3, but alsopentadecane and heptadecene. This result indicates that alr5283 in E.coli converts hexadecanal and octadecenal to pentadecane andheptadecene, respectively, and therefore is an active fatty aldehydedecarbonylase.

Example 9 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andAcaryochloris marina MBIC11017 AM1_4041

The genomic DNA encoding Acaryochloris marina MBIC11017 AM1_4041(YP_001518340; putative decarbonylase) (SEQ ID NO:9) was codon optimizedfor expression in E. coli (SEQ ID NO:46), synthesized, and cloned intothe NdeI and XhoI sites of vector OP-183 (pACYC derivative) under thecontrol of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example26.

As shown in FIG. 12, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and A. marina MBIC11017 AM1_4041-bearing vectors producedthe same fatty aldehydes and fatty alcohols as in Example 3, but alsotridecane, pentadecene, pentadecane and heptadecene. This resultindicates that AM1_4041 in E. coli converts tetradecanal, hexadecenal,hexadecanal and octadecenal to tridecane, pentadecene, pentadecane andheptadecene, respectively, and therefore is an active fatty aldehydedecarbonylase.

Example 10 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andThermosynechococcus elongatus BP-1 tll1313

The genomic DNA encoding Thermosynechococcus elongatus BP-1 tll1313(NP_682103; putative decarbonylase) (SEQ ID NO:11) was codon optimizedfor expression in E. coli (SEQ ID NO:47), synthesized, and cloned intothe NdeI and XhoI sites of vector OP-183 (pACYC derivative) under thecontrol of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example26.

As shown in FIG. 13, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and T. elongatus BP-1 tll1313-bearing vectors produced thesame fatty aldehydes and fatty alcohols as in Example 3, but alsopentadecane and heptadecene. This result indicates that tll1313 in E.coli converts hexadecanal and octadecenal to pentadecane andheptadecene, respectively, and therefore is an active fatty aldehydedecarbonylase.

Example 11 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andSynechococcus sp. JA-3-3Ab CYA_0415

The genomic DNA encoding Synechococcus sp. JA-3-3Ab CYA_0415 (YP_473897;putative decarbonylase) (SEQ ID NO:13) was codon optimized forexpression in E. coli (SEQ ID NO:48), synthesized, and cloned into theNdeI and XhoI sites of vector OP-183 (pACYC derivative) under thecontrol of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example26.

As shown in FIG. 14, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and Synechococcus sp. JA-3-3Ab CYA_0415-bearing vectorsproduced the same fatty aldehydes and fatty alcohols as in Example 3,but also pentadecane and heptadecene. This result indicates thatNpun02004178 in E. coli converts hexadecanal and octadecenal topentadecane and heptadecene, respectively, and therefore is an activefatty aldehyde decarbonylase.

Example 12 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andGloeobacter violaceus PCC7421 gll3146

The genomic DNA encoding Gloeobacter violaceus PCC7421 gll3146(NP_926092; putative decarbonylase) (SEQ ID NO:15) was amplified andcloned into the NdeI and XhoI sites of vector OP-183 (pACYC derivative)under the control of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example1.

As shown in FIG. 15, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and G. violaceus PCC7421 gll3146-bearing vectors producedthe same fatty aldehydes and fatty alcohols as in Example 3, but alsopentadecane and heptadecene. This result indicates that gll3146 in E.coli converts hexadecanal and octadecenal to pentadecane andheptadecene, respectively, and therefore is an active fatty aldehydedecarbonylase.

Example 13 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andProchlorococcus marinus MIT9313 PMT1231

The genomic DNA encoding Prochlorococcus marinus MIT9313 PMT1231(NP_895059; putative decarbonylase) (SEQ ID NO:17) was codon optimizedfor expression in E. coli (SEQ ID NO:49), synthesized, and cloned intothe NdeI and XhoI sites of vector OP-183 (pACYC derivative) under thecontrol of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example26.

As shown in FIG. 16, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and P. marinus MIT9313 PMT1231-bearing vectors produced thesame fatty aldehydes and fatty alcohols as in Example 3, but alsopentadecane and heptadecene. This result indicates that PMT1231 in E.coli converts hexadecanal and octadecenal to pentadecane andheptadecene, respectively, and therefore is an active fatty aldehydedecarbonylase.

Example 14 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andProchlorococcus marinus CCMP1986 PMM0532

The genomic DNA encoding Prochlorococcus marinus CCMP1986 PMM0532(NP_892650; putative decarbonylase) (SEQ ID NO:19) was amplified andcloned into the NdeI and XhoI sites of vector OP-183 (pACYC derivative)under the control of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example1.

As shown in FIG. 17, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and P. marinus CCMP1986 PMM0532-bearing vectors producedthe same fatty aldehydes and fatty alcohols as in Example 3, but alsopentadecane and heptadecene. This result indicates that PMM0532 in E.coli converts hexadecanal and octadecenal to pentadecane andheptadecene, respectively, and therefore is an active fatty aldehydedecarbonylase.

Example 15 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andProchlorococcus mariunus NATL2A PMN2A_1863

The genomic DNA encoding Prochlorococcus mariunus NATL2A PMN2A_1863(YP_293054; putative decarbonylase) (SEQ ID NO:21) was codon optimizedfor expression in E. coli (SEQ ID NO:51), synthesized, and cloned intothe NdeI and XhoI sites of vector OP-183 (pACYC derivative) under thecontrol of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example26.

As shown in FIG. 18, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and P. mariunus NATL2A PMN2A_1863-bearing vectors producedthe same fatty aldehydes and fatty alcohols as in Example 3, but alsopentadecane and heptadecene. This result indicates that PMN2A_1863 in E.coli converts hexadecanal and octadecenal to pentadecane andheptadecene, respectively, and therefore is an active fatty aldehydedecarbonylase.

Example 16 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andSynechococcus sp. RS9917 RS9917_09941

The genomic DNA encoding Synechococcus sp. RS9917 RS9917_09941(ZP_01079772; putative decarbonylase) (SEQ ID NO:23) was codon optimizedfor expression in E. coli (SEQ ID NO:52), synthesized, and cloned intothe NdeI and XhoI sites of vector OP-183 (pACYC derivative) under thecontrol of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example26.

As shown in FIG. 19, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and Synechococcus sp. RS9917 RS9917_09941-bearing vectorsproduced the same fatty aldehydes and fatty alcohols as in Example 3,but also pentadecane and heptadecene. This result indicates thatRS9917_09941 in E. coli converts hexadecanal and octadecenal topentadecane and heptadecene, respectively, and therefore is an activefatty aldehyde decarbonylase.

Example 17 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andSynechococcus sp. RS9917 RS9917_12945

The genomic DNA encoding Synechococcus sp. RS9917 RS9917_12945(ZP_01080370; putative decarbonylase) (SEQ ID NO:25) was codon optimizedfor expression in E. coli (SEQ ID NO:53), synthesized, and cloned intothe NdeI and XhoI sites of vector OP-183 (pACYC derivative) under thecontrol of the P_(trc) promoter. The resulting construct wascotransformed with OP80-PCC7942_1594 into E. coli MG1655 and the cellswere grown at 37° C. in M9 minimal media supplemented with 100 μg/mLspectinomycin and 100 μg/mL carbenicillin. The cells were cultured andextracted as in Example 3 and analyzed by GC-MS as described in Example26.

As shown in FIG. 20, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and Synechococcus sp. RS9917 RS9917_12945-bearing vectorsproduced the same fatty aldehydes and fatty alcohols as in Example 3,but also pentadecane and heptadecene. This result indicates thatRS9917_12945 in E. coli converts hexadecanal and octadecenal topentadecane and heptadecene, respectively, and therefore is an activefatty aldehyde decarbonylase.

Example 18 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andCyanothece sp. ATCC51142 cce_0778

The genomic DNA encoding Cyanothece sp. ATCC51142 cce_0778(YP_001802195; putative decarbonylase) (SEQ ID NO:27) was synthesizedand cloned into the NdeI and XhoI sites of vector OP-183 (pACYCderivative) under the control of the P_(trc) promoter. The resultingconstruct was cotransformed with OP80-PCC7942_1594 into E. coli MG1655and the cells were grown at 37° C. in M9 minimal media supplemented with100 μg/mL spectinomycin and 100 μg/mL carbenicillin. The cells werecultured and extracted as in Example 3 and analyzed by GC-MS asdescribed in Example 26.

As shown in FIG. 21, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and Cyanothece sp. ATCC51142 cce_0778-bearing vectorsproduced the same fatty aldehydes and fatty alcohols as in Example 3,but also tridecane, pentadecene, pentadecane and heptadecene. Thisresult indicates that ATCC51142 cce_0778 in E. coli convertstetradecanal, hexadecenal, hexadecanal and octadecenal to tridecane,pentadecene, pentadecane and heptadecene, respectively, and therefore isan active fatty aldehyde decarbonylase.

Example 19 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andCyanothece sp. PCC7425 Cyan7425_0398

The genomic DNA encoding Cyanothece sp. PCC7425 Cyan7425_0398(YP_002481151; putative decarbonylase) (SEQ ID NO:29) was synthesizedand cloned into the NdeI and XhoI sites of vector OP-183 (pACYCderivative) under the control of the P_(trc) promoter. The resultingconstruct was cotransformed with OP80-PCC7942_1594 into E. coli MG1655and the cells were grown at 37° C. in M9 minimal media supplemented with100 μg/mL spectinomycin and 100 μg/mL carbenicillin. The cells werecultured and extracted as in Example 3 and analyzed by GC-MS asdescribed in Example 26.

As shown in FIG. 22, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and Cyanothece sp. PCC7425 Cyan7425_0398-bearing vectorsproduced the same fatty aldehydes and fatty alcohols as in Example 3,but also tridecane, pentadecene, pentadecane and heptadecene. Thisresult indicates that Cyan7425_0398 in E. coli converts tetradecanal,hexadecenal, hexadecanal and octadecenal to tridecane, pentadecene,pentadecane and heptadecene, respectively, and therefore is an activefatty aldehyde decarbonylase.

Example 20 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Synechococcus elongatus PCC7942 orf1594 andCyanothece sp. PCC7425 Cyan7425_2986

The genomic DNA encoding Cyanothece sp. PCC7425 Cyan7425_2986(YP_002483683; putative decarbonylase) (SEQ ID NO:31) was synthesizedand cloned into the NdeI and XhoI sites of vector OP-183 (pACYCderivative) under the control of the P_(trc) promoter. The resultingconstruct was cotransformed with OP80-PCC7942_1594 into E. coli MG1655and the cells were grown at 37° C. in M9 minimal media supplemented with100 μg/mL spectinomycin and 100 μg/mL carbenicillin. The cells werecultured and extracted as in Example 3 and analyzed by GC-MS asdescribed in Example 26.

As shown in FIG. 23, E. coli cells cotransformed with the S. elongatusPCC7942_1594 and Cyanothece sp. PCC7425 Cyan7425_2986-bearing vectorsproduced the same fatty aldehydes and fatty alcohols as in Example 3,but also tridecane, pentadecene, pentadecane and heptadecene. Thisresult indicates that Cyan7425_2986 in E. coli converts tetradecanal,hexadecenal, hexadecanal and octadecenal to tridecane, pentadecene,pentadecane and heptadecene, respectively, and therefore is an activefatty aldehyde decarbonylase.

Example 21 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Prochlorococcus marinus CCMP1986 PMM0533 andProchlorococcus mariunus CCMP1986 PMM0532

The genomic DNA encoding P. mariunus CCMP1986 PMM0533 (NP_892651;putative aldehyde-generating enzyme) (SEQ ID NO:71) and Prochlorococcusmariunus CCMP1986 PMM0532 (NP_892650; putative decarbonylase) (SEQ IDNO:19) were amplified and cloned into the NcoI and EcoRI sites of vectorOP-80 and the NdeI and XhoI sites of vector OP-183, respectively. Theresulting constructs were separately transformed and cotransformed intoE. coli MG1655 and the cells were grown at 37° C. in M9 minimal mediasupplemented with 100 μg/mL spectinomycin and 100 μg/mL carbenicillin.The cells were cultured and extracted as in Example 3 and analyzed byGC-MS as described in Example 26.

As shown in FIG. 24A, E. coli cells transformed with only the P.mariunus CCMP1986 PMM0533-bearing vector did not produce any fattyaldehydes or fatty alcohols. However, E. coli cells cotransformed withPMM0533 and PMM0532-bearing vectors produced hexadecanol, pentadecaneand heptadecene (FIG. 24B). This result indicates that PMM0533 onlyprovides fatty aldehyde substrates for the decarbonylation reaction whenit interacts with a decarbonylase, such as PMM0532.

Example 22 Production of Alkanes and Alkenes in a FattyAcyl-CoA-Producing E. coli Strain Through Heterologous Expression ofSynechococcus elongatus PCC7942 orf1594 and Acaryochloris marinaMBIC11017 AM1_4041

The genomic DNA encoding Acaryochloris marina MBIC11017 AM1_4041(YP_001518340; putative fatty aldehyde decarbonylase) (SEQ ID NO:9) wassynthesized and cloned into the NdeI and XhoI sites of vector OP-183(pACYC derivative) under the control of the P_(trc) promoter. Theresulting construct was cotransformed with OP80-PCC7942_1594 into E.coli MG1655 ΔfadE lacZ::P_(trc) ′tesA-fadD. This strain expresses acytoplasmic version of the E. coli thioesterase, ′TesA, and the E. coliacyl-CoA synthetase, FadD, under the control of the P_(trc) promoter,and therefore produces fatty acyl-CoAs. The cells were grown at 37° C.in M9 minimal media supplemented with 100 μg/mL spectinomycin and 100μg/mL carbenicillin. The cells were cultured and extracted as in Example3 and analyzed by GC-MS as described in Example 1.

As shown in FIG. 25, these E. coli cells cotransformed with S. elongatusPCC7942_1594 and A. marina MBIC11017 AM1_4041 also produced alkanes andfatty alcohols. This result indicates that S. elongatus PCC7942_1594 isable to use acyl-CoA as a substrate to produce hexadecenal, hexadecanaland octadecenal, which is then converted into pentadecene, pentadecaneand heptadecene, respectively, by A. marina MBIC11017 AM1_4041.

Example 23 Production of Alkanes and Alkenes in a FattyAcyl-CoA-Producing E. coli Strain Through Heterologous Expression ofSynechocystis sp. PCC6803 sll0209 and Synechocystis sp. PCC6803 sll0208

The genomic DNA encoding Synechocystis sp. PCC6803 sll0208 (NP_442147;putative fatty aldehyde decarbonylase) (SEQ ID NO:3) was synthesized andcloned into the NdeI and XhoI sites of vector OP-183 (pACYC derivative)under the control of the P_(trc) promoter. The genomic DNA encodingSynechocystis sp. PCC6803 sll0209 (NP_442146; acyl-ACP reductase) (SEQID NO:67) was synthesized and cloned into the NcoI and EcoRI sites ofvector OP-183 (pACYC derivative) under the control of the P_(trc)promoter. The resulting constructs were cotransformed with into E. coliMG1655 ΔfadE lacZ::P_(trc) ′tesA-fadD. This strain expresses acytoplasmic version of the E. coli thioesterase, ′TesA, and the E. coliacyl-CoA synthetase, FadD, under the control of the P_(trc) promoter,and therefore produces fatty acyl-CoAs. The cells were grown at 37° C.in M9 minimal media supplemented with 100 μg/mL spectinomycin and 100μg/mL carbenicillin. The cells were cultured and extracted as in Example3 and analyzed by GC-MS as described in Example 26.

As shown in FIG. 26, these E. coli cells transformed with Synechocystissp. PCC6803 sll0209 did not produce any fatty aldehydes or fattyalcohols. However, when cotransformed with Synechocystis sp. PCC6803sll0208 and sll0209, they produced alkanes, fatty aldehydes and fattyalcohols. This result indicates that Synechocystis sp. PCC6803 sll0209is able to use acyl-CoA as a substrate to produce fatty aldehydes suchas tetradecanal, hexadecanal and octadecenal, but only when coexpressedwith a fatty aldehyde decarbonylase. The fatty aldehydes apparently arefurther reduced to the corresponding fatty alcohols, tetradecanol,hexadecanol and octadecenol, by an endogenous E. coli aldehyde reductaseactivity. In this experiment, octadecenal was converted into heptadeceneby Synechocystis sp. PCC6803 sll0208.

Example 24 Production of Alkanes and Alkenes in a FattyAldehyde-Producing E. coli Strain Through Heterologous Expression ofNostoc punctiforme PCC73102 Npun02004178 and Several of its Homologs

The genomic DNA encoding Nostoc punctiforme PCC73102 Npun02004178(ZP_00108838; putative fatty aldehyde decarbonylase) (SEQ ID NO:5) wasamplified and cloned into the NdeI and XhoI sites of vector OP-183(pACYC derivative) under the control of the P_(trc) promoter. Thegenomic DNA encoding Mycobacterium smegmatis strain MC2 155 orfMSMEG_5739 (YP_889972, putative carboxylic acid reductase) (SEQ IDNO:85) was amplified and cloned into the NcoI and EcoRI sites of vectorOP-180 (pCL1920 derivative) under the control of the P_(trc) promoter.The two resulting constructs were cotransformed into E. coli MG1655ΔfadD lacZ::P_(trc)-′tesA. In this strain, fatty aldehydes were providedby MSMEG_5739, which reduces free fatty acids (formed by the action of′TesA) to fatty aldehydes. The cells were grown at 37° C. in M9 minimalmedia supplemented with 100 μg/mL spectinomycin and 100 μg/mLcarbenicillin. The cells were cultured and extracted as in Example 3 andanalyzed by GC-MS as described in Example 1.

As shown in FIG. 27, these E. coli cells cotransformed with the N.punctiforme PCC73102 Npun02004178 and M. smegmatis strain MC2 155MSMEG_5739-bearing vectors produced tridecane, pentadecene andpentadecane. This result indicates that Npun02004178 in E. coli convertstetradecanal, hexadecenal and hexadecanal provided by the carboxylicacid reductase MSMEG_5739 to tridecane, pentadecene and pentadecane. Asshown in FIG. 28, in the same experimental set-up, the following fattyaldehyde decarbonylases also converted fatty aldehydes provided byMSMEG_5739 to the corresponding alkanes when expressed in E. coli MG1655ΔfadD Nostoc sp. PCC7210 alr5283 (SEQ ID NO:7), P. mariunus CCMP1986PMM0532 (SEQ ID NO:19), G. violaceus PCC7421 gll3146 (SEQ ID NO:15),Synechococcus sp. RS9917_09941 (SEQ ID NO:23), Synechococcus sp.RS9917_12945 (SEQ ID NO:25), and A. marina MBIC11017 AM1_4041 (SEQ IDNO:9).

Example 25 Cyanobacterial Fatty Aldehyde Decarbonylases Belong to theClass of Non-Heme Diiron Proteins. Site-Directed Mutagenesis ofConserved Histidines to Phenylalanines in Nostoc punctiforme PCC73102Npun02004178 Does Not Abolish its Catalytic Function

As discussed in Example 13, the hypothetical protein PMT1231 fromProchlorococcus marinus MIT9313 (SEQ ID NO:18) is an active fattyaldehyde decarbonylase. Based on the three-dimensional structure ofPMT1231, which is available at 1.8 Å resolution (pdb2OC5A) (see FIG.29B), cyanobacterial fatty aldehyde decarbonylases have structuralsimilarity with non-heme diiron proteins, in particular with class Iribonuclease reductase subunit β proteins, RNRβ (Stubbe andRiggs-Gelasco, TIBS 1998, vol. 23, pp. 438) (see FIG. 29A). Class Ia andIb RNRβ contains a diferric tyrosyl radical that mediates the catalyticactivity of RNRα (reduction of ribonucleotides to deoxyribonucleotides).In E. coli RNRβ, this tyrosine is in position 122 and is in closeproximity to one of the active site's iron molecules. Structuralalignment showed that PMT1231 contained a phenylalanine in the sameposition as RNRb tyr122, suggesting a different catalytic mechanism forcyanobacterial fatty aldehyde decarbonylases. However, an alignment ofall decarbonylases showed that two tyrosine residues were completelyconserved in all sequences, tyr135 and tyr138 with respect to PMT1231,with tyr135 being in close proximity (5.5 Å) to one of the active siteiron molecules (see FIG. 29C). To examine whether either of the twoconserved tyrosine residues is involved in the catalytic mechanism ofcyanobacterial fatty aldehyde decarbonylases, these residues werereplaced with phenylalanine in Npun02004178 (tyr 123 and tyr126) asfollows.

The genomic DNA encoding S. elongatus PCC7942 ORF1594 (SEQ ID NO:65) wascloned into the NcoI and EcoRI sites of vector OP-80 (pCL1920derivative) under the control of the P_(trc) promoter. The genomic DNAencoding N. punctiforme PCC73102 Npun02004178 (SEQ ID NO:5) was alsocloned into the NdeI and XhoI sites of vector OP-183 (pACYC177derivative) under the control of the P_(trc) promoter. The latterconstruct was used as a template to introduce a mutation at positions123 and 126 of the decarbonylase protein, changing the tyrosines tophenylalanines using the primersgttttgcgatcgcagcatttaacatttacatccccgttgccgacg andgttttgcgatcgcagcatataacattttcatccccgttgccgacg, respectively. Theresulting constructs were then transformed into E. coli MG1655. Thecells were grown at 37° C. in M9 minimal media supplemented with 1%glucose (w/v), and 100 μg/mL carbenicillin and spectinomycin. The cellswere cultured and extracted as in Example 3.

As shown in FIG. 30, the two Npun02004178 Tyr to Phe protein variantswere active and produced alkanes when coexpressed with S. elongatusPCC7942 ORF1594. This result indicates that in contrast to class Ia andIb RNRβ proteins, the catalytic mechanism of fatty aldehydedecarbonylases does not involve a tyrosyl radical.

Example 26 Biochemical Characterization of Nostoc punctiforme PCC73102Npun02004178

The genomic DNA encoding N. punctiforme PCC73102 Npun02004178 (SEQ IDNO:5) was cloned into the NdeI and XhoI sites of vector pET-15b underthe control of the T7 promoter. The resulting Npun02004178 proteincontained an N-terminal His-tag. An E. coli BL21 strain (DE3)(Invitrogen) was transformed with the plasmid by routine chemicaltransformation techniques. Protein expression was carried out by firstinoculating a colony of the E. coli strain in 5 mL of LB mediasupplemented with 100 mg/L of carbenicillin and shaken overnight at 37°C. to produce a starter culture. This starter cultures was used toinoculate 0.5 L of LB media supplemented with 100 mg/L of carbenicillin.The culture was shaken at 37° C. until an OD₆₀₀ value of 0.8 wasreached, and then IPTG was added to a final concentration of 1 mM. Theculture was then shaken at 37° C. for approximately 3 additional h. Theculture was then centrifuged at 3,700 rpm for 20 min at 4° C. The pelletwas then resuspended in 10 mL of buffer containing 100 mM sodiumphosphate buffer at pH 7.2 supplemented with Bacterial ProteaseArrest(GBiosciences). The cells were then sonicated at 12 W on ice for 9 swith 1.5 s of sonication followed by 1.5 s of rest. This procedure wasrepeated 5 times with one min intervals between each sonication cycle.The cell free extract was centrifuged at 10,000 rpm for 30 min at 4° C.5 mL of Ni-NTA (Qiagen) was added to the supernatant and the mixture wasgently stirred at 4° C. The slurry was passed over a column removing theresin from the lysate. The resin was then washed with 30 mL of buffercontaining 100 mM sodium phosphate buffer at pH 7.2 plus 30 mMimidazole. Finally, the protein was eluted with 10 mL of 100 mM sodiumphosphate buffer at pH 7.2 plus 250 mM imidazole. The protein solutionwas dialyzed with 200 volumes of 100 mM sodium phosphate buffer at pH7.2 with 20% glycerol. Protein concentration was determined using theBradford assay (Biorad). 5.6 mg/mL of Npun02004178 protein was obtained.

To synthesize octadecanal for the decarbonylase reaction, 500 mg ofoctadecanol (Sigma) was dissolved in 25 mL of dichloromethane. Next, 200mg of pyridinium chlorochromate (TCI America) was added to the solutionand stirred overnight. The reaction mixture was dried under vacuum toremove the dichloromethane. The remaining products were resuspended inhexane and filtered through Whatman filter paper. The filtrate was thendried under vacuum and resuspended in 5 mL of hexane and purified bysilica flash chromatography. The mixture was loaded onto the gravity fedcolumn in hexane and then washed with two column volumes of hexane. Theoctadecanal was then eluted with an 8:1 mixture of hexane and ethylacetate. Fractions containing octadecanal were pooled and analyzed usingthe GC/MS methods described below. The final product was 95% pure asdetermined by this method.

To test Npun02004178 protein for decarbonylation activity, the followingenzyme assays were set-up. 200 μL reactions were set up in 100 mM sodiumphosphate buffer at pH 7.2 with the following components at theirrespective final concentrations: 30 μM of purified Npun02004178 protein,200 μM octadecanal, 0.11 μg/mL spinach ferredoxin (Sigma), 0.05 units/mLspinach ferredoxin reductase (Sigma), and 1 mM NADPH (Sigma). Negativecontrols included the above reaction without Npun02004178, the abovereaction without octadecanal, and the above reaction without spinachferredoxin, ferredoxin reductase and NADPH. Each reaction was incubatedat 37° C. for 2 h before being extracted with 100 μL ethyl acetate.Samples were analyzed by GC/MS using the following parameters: run time:13.13 min; column: HP-5-MS Part No. 19091S-433E (length of 30 meters;I.D.: 0.25 mm narrowbore; film: 0.25 ìM); inject: 1 ìl Agilent 6850inlet; inlet: 300 C splitless; carrier gas: helium; flow: 1.3 mL/min;oven temp: 75° C. hold 5 min, 320 at 40° C./min, 320 hold 2 min; det:Agilent 5975B VL MSD; det. temp: 330° C.; scan: 50-550 M/Z. Heptadecanefrom Sigma was used as an authentic reference for determining compoundretention time and fragmentation pattern.

As shown in FIG. 31, in-vitro conversion of octadecanal to heptadecanewas observed in the presence of Npun02004178. The enzymaticdecarbonylation of octadecanal by Npun02004178 was dependent on theaddition of spinach ferredoxin reducatase, ferredoxin and NADPH.

Next, it was determined whether cyanobaterial ferredoxins and ferredoxinreductases can replace the spinach proteins in the in-vitro fattyaldehyde decarbonylase assay. The following four genes were clonedseparately into the NdeI and XhoI sites of pET-15b: N. punctiformePCC73102 Npun02003626 (ZP_00109192, ferredoxin oxidoreductase petHwithout the n-terminal allophycocyanin linker domain) (SEQ ID NO:87), N.punctiforme PCC73102 Npun02001001 (ZP_00111633, ferredoxin 1) (SEQ IDNO:89), N. punctiforme PCC73102 Npun02003530 (ZP_00109422, ferredoxin 2)(SEQ ID NO:91) and N. punctiforme PCC73102 Npun02003123 (ZP_00109501,ferredoxin 3) (SEQ ID NO:93). The four proteins were expressed andpurified as described above. 1 mg/mL of each ferredoxin and 4 mg/mL ofthe ferredoxin oxidoreductase was obtained. The three cyanobacterialferredoxins were tested with the cyanobacterial ferredoxinoxidoreductase using the enzymatic set-up described earlier with thefollowing changes. The final concentration of the ferredoxin reductasewas 60 μg/mL and the ferredoxins were at 50 μg/mL. The extractedenzymatic reactions were by GC/MS using the following parameters: runtime: 6.33 min; column: J&W 122-5711 DB-5ht (length of 15 meters; I.D.:0.25 mm narrowbore; film: 0.10 μM); inject: 1 μL Agilent 6850 inlet;inlet: 300° C. splitless; carrier gas: helium; flow: 1.3 mL/min; oventemp: 100° C. hold 0.5 min, 260 at 30° C./min, 260 hold 0.5 min; det:Agilent 5975B VL MSD; det. temp: 230° C.; scan: 50-550 M/Z.

As shown in FIG. 32, Npun02004178-dependent in-vitro conversion ofoctadecanal to heptadecane was observed in the presence of NADPH and thecyanobacterial ferredoxin oxidoreductase and any of the threecyanobacterial ferredoxins.

Example 27 Biochemical Characterization of Synechococcus elongatusPCC7942 orf1594

The genomic DNA encoding S. elongatus PCC7492 orf1594 (SEQ ID NO:65) wascloned into the NcoI and XhoI sites of vector pET-28b under the controlof the T7 promoter. The resulting PCC7942_orf1594 protein contained aC-terminal His-tag. An E. coli BL21 strain (DE3) (Invitrogen) wastransformed with the plasmid and PCC7942_orf1594 protein was expressedand purified as described in Example 22. The protein solution was storedin the following buffer: 50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 1mM THP, 10% glycerol. Protein concentration was determined using theBradford assay (Biorad). 2 mg/mL of PCC7942_orf1594 protein wasobtained.

To test PCC7942_orf1594 protein for acyl-ACP or acyl-CoA reductaseactivity, the following enzyme assays were set-up. 100 μL reactions wereset-up in 50 mM Tris-HCl buffer at pH 7.5 with the following componentsat their respective final concentrations: 10 μM of purifiedPCC7942_orf1594 protein, 0.01-1 mM acyl-CoA or acyl-ACP, 2 mM MgCl₂,0.2-2 mM NADPH. The reactions were incubated for 1 h at 37° C. and wherestopped by adding 100 μL ethyl acetate (containing 5 mg/l 1-octadeceneas internal standard). Samples were vortexed for 15 min and centrifugedat max speed for 3 min for phase separation. 80 μL of the top layer weretransferred into GC glass vials and analyzed by GC/MS as described inExample 26. The amount of aldehyde formed was calculated based on theinternal standard.

As shown in FIG. 33, PCC7942_orf1594 was able to reduce octadecanoyl-CoAto octadecanal. Reductase activity required divalent cations such asMg²⁺, Mn²⁺ or Fe²⁺ and NADPH as electron donor. NADH did not supportreductase activity. PCC7942_orf1594 was also able to reduceoctadecenoyl-CoA and octadecenoyl-ACP to octadecenal. The K_(m) valuesfor the reduction of octadecanoyl-CoA, octadecenoyl-CoA andoctadecenoyl-ACP in the presence of 2 mM NADPH were determined as 45±20μM, 82±22 μM and 7.8±2 μM, respectively. These results demonstrate thatPCC7942_orf1594, in vitro, reduces both acyl-CoAs and acyl-ACPs and thatthe enzyme apparently has a higher affinity for acyl-ACPs as compared toacyl-CoAs. The K_(m) value for NADPH in the presence of 0.5 mMoctadecanoyl-CoA for PCC7942_orf1594 was determined as 400±80 μM.

Next, the stereospecific hydride transfer from NADPH to a fatty aldehydecatalyzed by PCC7942_orf1594 was examined. Deutero-NADPH was preparedaccording to the following protocol. 5 mg of NADP and 3.6 mg ofD-glucose-1-d was added to 2.5 mL of 50 mM sodium phosphate buffer (pH7.0). Enzymatic production of labeled NADPH was initiated by theaddition of 5 units of glucose dehydrogenase from either Bacillusmegaterium (USB Corporation) for the production of R-(4-²H)NADPH orThermoplasma acidophilum (Sigma) for the production of S-(4-²H)NADPH.The reaction was incubated for 15 min at 37° C., centrifuge-filteredusing a 10 KDa MWCO Amicon Ultra centrifuge filter (Millipore), flashfrozen on dry ice, and stored at −80° C.

The in vitro assay reaction contained 50 mM Tris-HCl (pH 7.5), 10 μM ofpurified PCC7942_orf1594 protein, 1 mM octadecanoyl-CoA, 2 mM MgCl₂, and50 μL deutero-NADPH (prepared as described above) in a total volume of100 μL. After a 1 h incubation, the product of the enzymatic reactionwas extracted and analyzed as described above. The resulting fattyaldehyde detected by GC/MS was octadecanal (see FIG. 34). Becausehydride transfer from NADPH is stereospecific, both R-(4-²H)NADPH andS-(4-²H)NADPH were synthesized. Octadecanal with a plus one unit masswas observed using only the S-(4-²H)NADPH. The fact that the fattyaldehyde was labeled indicates that the deuterated hydrogen has beentransferred from the labeled NADPH to the labeled fatty aldehyde. Thisdemonstrates that NADPH is used in this enzymatic reaction and that thehydride transfer catalyzed by PCC7942_orf1594 is stereospecific.

Example 28 Intracellular and Extracellular Production of Fatty Aldehydesand Fatty Alcohols in E. coli Through Heterologous Expression ofSynechococcus elongatus PCC7942 orf1594

The genomic DNA encoding Synechococcus elongatus PCC7942 orf1594(YP_400611; acyl-ACP reductase) (SEQ ID NO:65) was amplified and clonedinto the NcoI and EcoRI sites of vector OP-80 (pCL1920 derivative) underthe control of the P_(trc) promoter. The resulting construct wascotransformed into E. coli MG1655 ΔfadE and the cells were grown at 37°C. in 15 mL Che-9 minimal media with 3% (w/v) glucose as carbon sourceand supplemented with 100 μg/mL spectinomycin and carbenicillin,respectively. When the culture reached OD₆₀₀ of 0.8-1.0, it was inducedwith 1 mM IPTG and cells were grown for an additional 24-48 h at 37° C.Che-9 minimal medium is defined as: 6 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/LNaCl, 2 g/L NH₄Cl, 0.25 g/L MgSO₄×7 H₂O, 11 mg/L CaCl₂, 27 mg/L Fe₃Cl×6H₂O, 2 mg/L ZnCl×4 H₂O, 2 mg/L Na₂MoO₄×2 H₂O, 1.9 mg/L CuSO₄×5 H₂O, 0.5mg/L H₃BO₃, 1 mg/L thiamine, 200 mM Bis-Tris (pH 7.25) and 0.1% (v/v)Triton-X100. When the culture reached OD₆₀₀ of 1.0-1.2, it was inducedwith 1 mM IPTG and cells were allowed to grow for an additional 40 hrsat 37° C. Cells from 0.5 mL of culture were extracted with 0.5 mL ofethyl acetate for total hydrocarbon production as described in Example26. Additionally, cells and supernatant were separated by centrifugation(4,000 g at RT for 10 min) and extracted separately.

The culture produced 620 mg/L fatty aldehydes (tetradecanal,heptadecenal, heptadecanal and octadecenal) and 1670 mg/L fatty alcohols(dodecanol, tetradecenol, tetradecanol, heptadecenol, heptadecanol andoctadecenol). FIG. 35 shows the chromatogram of the extractedsupernatant. It was determined that 73% of the fatty aldehydes and fattyalcohols were in the cell-free supernatant.

Example 29 Intracellular and Extracellular Production of Alkanes andAlkenes in E. coli Through Heterologous Expression of Synechococcuselongatus PCC7942 orf1594 and Nostoc punctiforme PCC73102 Npun02004178

The genomic DNA encoding Synechococcus elongatus PCC7942 orf1594(YP_400611; acyl-ACP reductase) (SEQ ID NO:65) was amplified and clonedinto the NcoI and EcoRI sites of vector OP-80 (pCL1920 derivative) underthe control of the P_(trc) promoter. The genomic DNA encoding Nostocpunctiforme PCC73102 Npun02004178 (ZP_00108838; fatty aldehydedecarbonylase) (SEQ ID NO:5) was amplified and cloned into the NdeI andXhoI sites of vector OP-183 (pACYC derivative) under the control of theP_(trc) promoter. The resulting constructs were cotransformed into E.coli MG1655 ΔfadE and the cells were grown at 37° C. in 15 mL Che9minimal media with 3% (w/v) glucose as carbon source and supplementedwith 100 μg/mL spectinomycin and carbenicillin, respectively. The cellswere grown, separated from the broth, extracted, and analyzed asdescribed in Example 28.

The culture produced 323 mg/L alkanes and alkenes (tridecane,pentadecene, pentadecane and heptadecene), 367 mg/L fatty aldehydes(tetradecanal, heptadecenal, heptadecanal and octadecenal) and 819 mg/Lfatty alcohols (tetradecanol, heptadecenol, heptadecanol andoctadecenol). FIG. 36 shows the chromatogram of the extractedsupernatant. It was determined that 86% of the alkanes, alkenes, fattyaldehydes and fatty alcohols were in the cell-free supernatant.

Example 30 Production of Alkanes and Alkenes in E. coli ThroughHeterologous Expression of Nostoc sp. PCC7210 alr5284 and Nostoc sp.PCC7210 alr5283

The genomic DNA encoding Nostoc sp. PCC7210 alr5284 (NP_489324; putativealdehyde-generating enzyme) (SEQ ID NO:81) was amplified and cloned intothe NcoI and EcoRI sites of vector OP-80 (pCL1920 derivative) under thecontrol of the P_(trc) promoter. The genomic DNA encoding Nostoc sp.PCC7210 alr5283 (NP_489323; putative decarbonylase) (SEQ ID NO:7) wasamplified and cloned into the NdeI and XhoI sites of vector OP-183(pACYC derivative) under the control of the P_(trc) promoter. Theresulting constructs were cotransformed into E. coli MG1655 and thecells were grown at 37° C. in 15 mL Che9 minimal media with 3% (w/v)glucose as carbon source and supplemented with 100 μg/mL spectinomycinand carbenicillin, respectively (as described in Example 28). Cells from0.5 mL of culture were extracted and analyzed as described in Example 3and analyzed by GC-MS as described in Example 26.

As shown in FIG. 37, E. coli cells cotransformed with the Nostoc sp.PCC7210 alr5284 and Nostoc sp. PCC7210 alr5283-bearing vectors producedtridecane, pentadecene, pentadecane, tetradecanol and hexadecanol. Thisresult indicates that coexpression of Nostoc sp. PCC7210 alr5284 andalr5283 is sufficient for E. coli to produce fatty alcohols, alkanes andalkenes.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

Percent Organism (and Percent Sequence Accession Sequence Identity ofNumber) of Identity of Aldehyde Aldehyde Aldehyde Alkane/AlkeneAlkane/Alkene Organism (and Accession Production Example in BiosyntheticBiosynthetic Biosynthetic Biosynthetic Biosynthetic Number) ofAlkane/Alkene of Alkanes Specification Polypeptides PolypeptidesPolypeptides Polypeptides Polypeptides Biosynthetic Polypeptides andAlkenes  5 SEQ ID NO: 65 100% Synechococcus SEQ ID NO: 1 100%Synechococcus elongatus yes elongatus PCC7942 orf1593 PCC7942(YP_400610) orf1594 (YP_400611)  6 SEQ ID NO: 65 100% Synechococcus SEQID NO: 5  72% Nostoc punctiforme yes elongatus PCC73102 Npun02004178PCC7942 (ZP_00108838) orf1594 (YP_400611)  7 SEQ ID NO: 65 100%Synechococcus SEQ ID NO: 3  72% Synechocystis sp. PCC6803 yes elongatussll0208 (NP_442147) PCC7942 orf1594 (YP_400611)  8 SEQ ID NO: 65 100%Synechococcus SEQ ID NO: 7  74% Nostoc sp. PCC721_4893230 yes elongatusalr5283 (NP) PCC7942 orf1594 (YP_400611)  9 SEQ ID NO: 65 100%Synechococcus SEQ ID NO: 9  71% Acaryochloris marina yes elongatusMBIC11017 AM1_4041 PCC7942 (YP_001518340) orf1594 (YP_400611) 10 SEQ IDNO: 65 100% Synechococcus SEQ ID NO: 11  70% Thermosynechococcus yeselongatus elongatus BP-1 tll1313 PCC7942 (NP_682103) orf1594 (YP_400611)11 SEQ ID NO: 65 100% Synechococcus SEQ ID NO: 13  68% Synechococcus sp.JA-3-3Ab yes elongatus CYA_0415 (YP_473897) PCC7942 orf1594 (YP_400611)12 SEQ ID NO: 65 100% Synechococcus SEQ ID NO: 15  63% Gloeobacterviolaceus yes elongatus PCC7421 gll3146 PCC7942 (NP_926092) orf1594(YP_400611) 13 SEQ ID NO: 65 100% Synechococcus SEQ ID NO: 49  63%Prochlorococcus marinus yes elongatus MIT9313 PMT1231 PCC7942(NP_895059) (codon orf1594 optimized for E. coli) (YP_400611) 14 SEQ IDNO: 65 100% Synechococcus SEQ ID NO: 19  60% Prochlorococcus marinus yeselongatus CCMP1986 PMM0532 PCC7942 (NP_892650) orf1594 (YP_400611) 15SEQ ID NO: 65 100% Synechococcus SEQ ID NO: 51  59% Prochlorococcusmariunus yes elongatus NATL2A PMN2A_1863 PCC7942 (YP_293054) (codonorf1594 optimized for E. coli) (YP_400611) 16 SEQ ID NO: 65 100%Synechococcus SEQ ID NO: 52  68% Synechococcus sp. RS9917 yes elongatusRS9917_09941 PCC7942 (ZP_01079772) (codon orf1594 optimized for E. coli)(YP_400611) 17 SEQ ID NO: 65 100% Synechococcus SEQ ID NO: 53  43%Synechococcus sp. RS9917 yes elongatus RS9917_12945 PCC7942(ZP_01080370) (codon orf1594 optimized for E. coli) (YP_400611) 18 SEQID NO: 65 100% Synechococcus SEQ ID NO: 27  73% Cyanothece sp. ATCC51142yes elongatus cce_0778 (YP_001802195) PCC7942 orf1594 (YP_400611) 19 SEQID NO: 65 100% Synechococcus SEQ ID NO: 29  71% Cyanothece sp. PCC7425yes elongatus Cyan7425_0398 PCC7942 (YP_002481151) orf1594 (YP_400611)20 SEQ ID NO: 65 100% Synechococcus SEQ ID NO: 31  59% Cyanothece sp.PCC7425 yes elongatus Cyan7425_2986 PCC7942 (YP_002483683) orf1594(YP_400611) 21 SEQ ID NO: 71  63% P. mariunus SEQ ID NO: 19  60%Prochlorococcus mariunus yes CCMP1986 CCMP1986 PMM0532 PMM0533(NP_892650) (NP_892651) 22 SEQ ID NO: 65 100% Synechococcus SEQ ID NO: 9 71% Acaryochloris marina yes elongatus MBIC11017 AM1_4041 PCC7942(YP_001518340) orf1594 (YP_400611) 23 SEQ ID NO: 67  68% Synechocystissp. SEQ ID NO: 3  72% Synechocystis sp. PCC6803 yes PCC6803 sll0208(NP_442147) sll0209 (NP_442146) 29 SEQ ID NO: 65 100% Synechococcus SEQID NO: 5  72% Nostoc punctiforme yes elongatus PCC73102 Npun02004178PCC7942 (ZP_00108838) orf1594 (YP_400611) 30 SEQ ID NO: 81  72% Nostocsp. SEQ ID NO: 7  74% Nostoc sp. PCC7210 alr5283 yes PCC7210 (NP_489323)alr5284 (NP_489324)

What is claimed is:
 1. An engineered microorganism for production of analkane or alkene, comprising: (i) a first nucleotide sequence encodingan aldehyde biosynthetic polypeptide that catalyzes the conversion ofacyl ACP to a fatty aldehyde in an engineered microorganism, wherein thealdehyde biosynthetic polypeptide encoded by the first nucleotidesequence has at least 85% sequence identity to SEQ ID NO: 72; and (ii) asecond nucleotide sequence encoding an alkane or alkene biosyntheticpolypeptide that catalyzes the conversion of a fatty aldehyde to analkane or alkene in said engineered microorganism, wherein the alkane oralkene biosynthetic polypeptide encoded by the second nucleotidesequence has at least 85% sequence identity to SEQ ID NO: 20, andwherein each of said first and second nucleotide sequences is operablylinked to a promoter.
 2. The engineered microorganism of claim 1,wherein said engineered microorganism comprises aldehyde biosyntheticactivity.
 3. The engineered microorganism of claim 2, wherein saidengineered microorganism further comprises alkane biosynthetic activity.4. The engineered microorganism of claim 3, wherein said engineeredmicroorganism further comprises alkene biosynthetic activity.
 5. Theengineered microorganism of claim 1, wherein the engineeredmicroorganism further comprises ferredoxin and ferredoxin reductase. 6.The engineered microorganism of claim 1, wherein the engineeredmicroorganism is selected from the group consisting of a bacteria, ayeast, a fungi, and an algae.
 7. The engineered microorganism of claim6, wherein the bacteria is E. coli.
 8. The engineered microorganism ofclaim 6, wherein the bacteria is cyanobacteria.
 9. The engineeredmicroorganism of claim 6, wherein the fungi is a filamentous fungi. 10.A cell culture comprising the engineered microorganism of claim
 1. 11.The cell culture of claim 10, wherein the alkane or alkene comprises aC13 to C21 alkane or alkene.
 12. The cell culture of claim 11, whereinthe alkane or alkene is selected from the group consisting of tridecane,methyltridecane, nonadecane, methylnonadecane, heptadecane,methylheptadecane, pentadecane, methylpentadecane, pentadecene,heptadecene, methylpentadecene, and methylheptadecene.
 13. The cellculture of claim 11, wherein the alkane or alkene has a 8¹³C of about−15.4 or greater.
 14. The cell culture of claim 11, wherein the alkaneor alkene has an f_(M) ¹⁴C of at least about 1.003.
 15. A method ofproducing an alkane or alkene from an engineered microorganism, themethod comprising, (a) expressing a first nucleotide sequence encodingan aldehyde biosynthetic polypeptide that catalyzes the conversion ofacyl ACP to a fatty aldehyde in said engineered microorganism, whereinthe aldehyde biosynthetic polypeptide encoded by the first nucleotidesequence has at least 85% sequence identity to SEQ ID NO: 72; (b)expressing a second nucleotide sequence encoding an alkane or alkenebiosynthetic polypeptide that catalyzes the conversion of a fattyaldehyde to an alkane or alkene in said engineered microorganism,wherein the alkane or alkene biosynthetic polypeptide encoded by thesecond nucleotide sequence has at least 85% sequence identity to SEQ IDN0:20, each of said first and second nucleotide sequences is operablylinked to a promoter; and (c) culturing the engineered microorganism ina culture media containing a carbohydrate carbon source under conditionseffective to produce an alkane or alkene.
 16. The method of claim 15,wherein the engineered microorganism is selected from the groupconsisting of a bacteria, a yeast, a fungi, and an algae.
 17. The methodof claim 16, wherein the bacteria is E. coli.
 18. The method of claim16, wherein the bacteria is cyanobacteria.
 19. The method of claim 16,wherein the fungi is a filamentous fungi.
 20. The method of claim 15,wherein the alkane or alkene comprises a C₁₃ to C₂₁ alkane or alkene.21. The method of claim 20, wherein the alkane or alkene is selectedfrom the group consisting of tridecane, methyltridecane, nonadecane,methylnonadecane, heptadecane, methylheptadecane, pentadecane,methylpentadecane, pentadecene, heptadecene, methylpentadecene, andmethylheptadecene.
 22. The method of claim 20, wherein the alkane oralkene has a 8¹³C of about −15.4 or greater.
 23. The method of claim 20,wherein the alkane or alkene has an f_(M) ¹⁴C of at least about 1.003.